Stabilized bitamin c derivatives having a peptide molecule, preparation method thereof, and composition containing the same

ABSTRACT

The present invention relates to a stabilized vitamin C derivative with a peptide molecule linked to vitamin C or a pharmaceutically acceptable salt thereof, a method of preparing the same, and a composition containing the same.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to and the benefit of Korean PatentApplication No. 2006-0056580 filed on Jun. 22, 2006, which is herebyincorporated by reference for all purposes as if fully set forth herein.

BACKGROUND OF THE INVENTION

(a) Field of the Invention

The present invention relates to a stabilized vitamin C derivativehaving a peptide molecule or a pharmaceutically acceptable salt thereof,a preparation method of the vitamin C derivative, and a compositioncontaining the same.

(b) Description of the Related Art

Skin plays the roles of protection, barrier, body temperatureregulation, excretion, respiration, and so forth, and consists ofepidermis, dermis, and hypodermis. The epidermis is the thinnest layer,consisting of keratinocytes and melanocytes. The dermis accounts forabout 95% of skin and is the layer that takes up skin moisturization andprotection. It consists of collagen and elastin, which are proteinfibers that play important roles in skin elasticity (or wrinkles). Thedermis contains blood vessels, nerves, mast cells associated withallergic reactions, and natural moisturizing factors such as Na-PCA,hyaluronic acid, etc. The hypodermis supplies nutrients to the epidermisand the dermis, determines body shape, maintains body temperature,absorbs external impact, and protects the cells therebelow.

As skin ages, its functions decline rapidly because of endogenous orexogenous factors. As aging proceeds, the layers of the epidermis, thedermis, and the hypodermis become thin, and the collagen and elastinfibers become thinner and less elastic, resulting in wrinkles. Further,as the lipid composition and content in the lipid barrier that protectsthe skin changes, the moisture content in skin decreases, resulting indryness and other physiological changes. Furthermore, melasma, freckles,pigmentation, and various other skin lesions are induced.

In order to solve the skin aging-related problems, much research hasbeen carried out on various active substances effective in skinimprovement, and compositions containing them.

Antioxidant vitamins, such as vitamin C, vitamin E, β-carotene, and soon, have been used to inhibit skin aging. Among them, vitamin C is knownto be effective in improving skin wrinkles, improving pigmentationproblems such as melasma, freckles, dark spots, etc., and enhancing theimmune system through protection from UV, antioxidant activity, andpromotion of collagen formation. The functions of vitamin C in the bodyare as follows. Vitamin C effectively blocks UV, particularly UV A[Darr, D. et al., 1996, Acta Derm. Venereol. (Strckh). 76: 264-268;Black, H. S. et al., 1975, J. Invest. Dermatol. 65: 412-414]. Also,vitamin C protects the skin from damage induced by UV B. When vitamin Cis applied on the skins of pigs and humans and then UV B is irradiatedthereon, it prevented erythema and sunburn (Darr, D. et al., 1992, Brit.J. Dermatil. 127: 247-253; Murry, J. et al., 1991, J. Invest. Dermatol.96: 587).

Vitamin C acts as a strong antioxidant, which neutralizes the reactiveoxygen species (ROS) generated in skin, blood, and other tissues bychemical contaminations, smoking, and particularly, UV. This is becauseof the structural characteristics of the vitamin C, which can accept twoelectrons and be readily oxidized to dehydro-L-ascorbic acid. Vitamin Cis an important factor in the non-enzymatic antioxidant defense systemof skin. When present in high concentration, vitamin C neutralizes suchROS as singlet oxygens, superoxide anions, hydroxy radicals, etc.,before they oxidize or modify proteins, nucleic acids, cytoplasmicmembrane lipids or other body constituents (Buettner, G. R. et al. 1996.Cadenas, E., Packer, L., eds. Handbook of antioxidants. pp. 91-115).

When administered percutaneously to the stratum corneum, vitamin Cimproves skin gloss and skin color, reduces wrinkles, and improves skinelasticity (U.S. Pat. No. 4,983,382), because it promotes collagensynthesis. Hydroxyproline, which accounts for about 10% of collagenpolypeptides, is synthesized by proline hydroxylase, wherein vitamin Cacts as a cofactor of this enzyme (Tomita, Y. et al., 1980, J. Invest.Dermatol. 75(5): 379-382). That is, vitamin C activates prolinehydroxylase to promote the synthesis of hydroxyproline, which in turnpromotes the biosynthesis of the triple-helical collagen, therebyreducing skin wrinkles and improving skin condition.

Vitamin C offers a superior skin whitening effect, because it inhibitsthe activity of tyrosinase, which is important in producing melanin, andthe production of melanin (Tomita, Y. et al., 1980, J. Invset. Dermatol.75(5); 379-382).

In addition, vitamin C enhances the immune system by inhibiting therelease of allergy-inducing histamine from the cell membrane, therebypreventing allergic reactions. It has been proven that vitamin Cprotects mice exposed to antigens from UV-induced immune suppression andresistance (Nakamura, T. et al., 1997, J. Invest. Dermatol. 109: 20-24).Further, vitamin C helps the phagocytosis of leukocytes, facilitates themovements of leukocytes during infection to inhibit infection, promotesthe biosynthesis of interferon, which is a protein that suppresses theproliferation of viruses, and improves resistance to a variety ofinfectious diseases. Further, vitamin C also participates in folic acidmetabolism and amino acid metabolism.

Vitamin C is a water-soluble substance with the chemical formula C₆H₈O₆.Vitamin C is hydrophilic because of the hydroxyl groups present in C-2,C-3, C-5, and C-6 positions. In neutral pH, e.g., in water, the hydroxylgroups, particularly those in C-2 and C-3 positions, in Vitamin Cbecomes negatively charged, to be quickly and completely dissolved inaqueous solutions. However, under non-aqueous organic environments,e.g., in skin, vitamin C is not completely dissolved. Further, becausevitamin C is not readily dissolved in the organic solvents commonly usedin the external preparations, e.g., glycerin, propylene glycol, avariety of fats, etc., transdermal delivery of vitamin C is restricted.That is, in a non-ionized state, the pH should be maintained at 4.2 orlower in order for vitamin C to easily penetrate the skin barrier.

It is known that vitamin C is accumulated in skin 20 to 40 times higherwhen transdermally (externally) administered than orally administered.External preparations for improving wrinkles by UV blocking,antioxidation and promotion of collagen production, improvingpigmentation problems such as melasma, freckles, dark spots, etc., andenhancing the immune system, should have a high degree of transdermalabsorption because the active ingredients of the preparations need toreach the cells in the epidermis by passing through the stratum corneumof skin. In general, the degree of transdermal absorption of a substancerelates to its lipophilicity. It is known that a substance havinglipophilicity similar to that of skin is readily absorbed transdermally,because the partition coefficient into skin is high. In contrast,vitamin C is not readily absorbed transdermally because of highhydrophilicity and a low partition coefficient into skin.

There has been much research to improve safety, stability, andtransdermal absorptivity of vitamin C.

Several types of vitamin C derivatives have been suggested. The firsttype of derivative is a phosphated ascorbic acid or a metal salt ofphosphated ascorbic acid. This derivative is obtained by linking thehydroxyl group of the C-2 or C-3 position of ascorbic acid through anester bond with the phosphate to form ascorbyl-2-phosphate orascorbyl-3-phosphate, respectively. Compared with other derivatives,these derivatives are easily transited to L-ascorbic acid that can beutilized by the human body. However, they are not readily absorbedtransdermally because they have negative charges.

The second type of derivative is an ascorbic acid bonded to a fattyacid. U.S. Pat. No. 5,409,693 discloses a vitamin C derivative in theform of a fat-soluble fatty acid ester of ascorbic acid, such asascorbyl palmitate, ascorbyl laurate, ascorbyl myristate, ascorbylstearate, etc. Among them, ascorbyl-6-palmitate is most frequentlyutilized. Although ascorbyl-6-palmitate is readily absorbedtransdermally, it is not readily transited to L-ascorbic acid. It isreported that ascorbyl-6-palmitate failed in protecting the skin of micefrom photo-oxidation (Bissett, D. et al., 1990, PhotodermatolPhotoimmunol Photomed 7: 56-62). There is another report that thetreatment of skin with a serum containing 10 wt % ascorbyl-6-palmitatedid not result in a significant increase of ascorbic acid (Pinnell, S.R. et al., 2001. Dermatologic Surgery. 27(2): 137-142).

The third type of derivative is ascorbic acid derivatives having amonosaccharide, such as glycosylated, mannosylated, fructosylated,fucosylated, galactosylated, N-acetylglucosaminated, orN-acetylmuraminated form. However, the physiological activities of thesederivatives in the body have not yet been elucidated specifically andaccurately.

The fourth type of derivative is an ascorbic acid bonded with acollagen-producing peptide. Korean Patent No. 0459679 discloses aderivative obtained by linking the hydroxyl group of the C-5 or C-6position of ascorbic acid with a succinoyl group through an ester bond,and linking it with a collagen-producing peptide through an imide bond.This ascorbic acid derivative shows better efficiency than ascorbicacid, but is less stable. Collagen is frequently found in skin, bloodvessels, internal organs, bones, etc. It accounts for 70% of the dermisin skin. The fascia which surrounds muscles is made up of collagen.Collagen, which accounts about 30 wt % of the total proteins in thebody, offers mechanical solidity to skin, confers resistance and bindingstrength of connective tissues, supports cell adhesion, and induces celldivision and differentiation during growth or healing of wounds.Collagen is produced in fibroblasts. The amount of collagen decreaseswith natural aging and photo-aging. It is known that the amount ofcollagen decreases by 65% from the age of 20 to 80 (Shuster S., 1975,British Journal of Dermatology, 93(6): 639-643).

With the finding that activated collagen synthesis in the body increasesthe dermal matrix, resulting in the effects of improving wound healing,skin elasticity, wrinkle reduction, etc., collagen has been utilized incosmetics, foods, medicines, and so forth. Some oligopeptides havingless than 10 amino acids, which exist in collagen, are the smallestactivation unit. They relate to functions as messenger, stimulator, andneurotransmitter, and take part in such physiological metabolisms asgrowth control, nursing, immunity, digestion, blood pressure, andhealing. Particularly, peptides that are effective in skin regenerationare disclosed in French Patent No. 2,668,265, U.S. Pat. No. 4,665,054,WO91/3488, WO91/7431, and so forth. However, these peptides tend to formprecipitates, thereby greatly reducing product stability.

Thus, the development of vitamin C derivatives with improved safety andstability and superior skin permeability is needed.

SUMMARY OF THE INVENTION

To satisfy the needs, the present invention intends to provide a vitaminC derivative with improved safety and stability and superior skinpermeability by introducing a phosphate derivative and a peptidemolecule, particularly a collagen-producing peptide, to vitamin C.

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BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a comparison of collagen biosynthesis capabilities usingthe compound of the present invention, vitamin C, magnesium ascorbylphosphate, succinoyl ascorbyl pentapeptide, and palmitoyl pentapeptide.

FIG. 2 shows a comparison of cell toxicity of the compound of thepresent invention, vitamin C, magnesium ascorbyl phosphate, succinoylascorbyl pentapeptide, and palmitoyl pentapeptide.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A more complete appreciation of the invention, and many of the attendantadvantages thereof, will be readily apparent as the same becomes betterunderstood by reference to the following detailed description.

The present invention relates to a stabilized vitamin C. derivativehaving a peptide molecule or a pharmaceutically acceptable salt thereof,a preparation method of the vitamin C derivative, and a compositioncontaining the same.

The present invention relates to a stabilized vitamin C derivative inwhich a phosphoryl group or its derivative is introduced at the C-2position, and a peptide, preferably a collagen-producing peptide, isintroduced at the C-5 or C-6 position of vitamin C, or apharmaceutically acceptable salt thereof.

In one aspect, the present invention relates to a stabilized vitamin Cderivative with a peptide molecule, which is represented by thefollowing Chemical Formula 1, and a pharmaceutically acceptable saltthereof:

wherein

R1 and R2 are —OH or

and are different from each other;

X is —OC(O)(CH₂)mC(O)—;

is a natural or non-natural peptide molecule, in which identical ordifferent amino acid residues are linked through amide bonds, and theamino acid residues are selected from the group consisting of glycine,lysine, histidine, serine, proline, hydroxyproline, threonine, glutamicacid, methionine, glutamine, and arginine;

R is a side chain of the amino acid;

n is an integer between 3 and 10;

m is an integer between 2 and 5;

W is

or glucose; and

R3 is selected from the group consisting of —N(CH₃)₂, —N(CH₂CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂C₆H₅, and —OH.

In a specific embodiment of the present invention, the compoundrepresented by Chemical Formula 1 may be a compound represented by thefollowing Chemical Formula 1a, 1b, 1c, or 1d:

In a preferred embodiment of the present invention,

in Chemical Formula 1, 1a, 1b, 1c, or 1d is a peptide, in whichidentical or different amino acid residues selected from the groupconsisting of glycine, lysine, histidine, serine, proline,hydroxyproline, threonine, arginine, glutamine, methionine, and glutamicacid are linked through amide bonding;

R is a side chain of the amino acid residue; and

n is an integer between 3 and 7.

In a more preferred embodiment, the peptide

may be a tri-, tetra-, penta, or hepta-peptide, with n being an integerbetween 3 and 6, particularly a peptide selected from the groupconsisting of glycine-lysine-histidine, glycine-histidine-lysine,glycine-proline-hydroxyproline,lysine-threonine-threonine-lysine-serine, and glutamic acid-glutamicacid-methionine-glutamine-arginine-arginine.

X may be —OC(O)(CH₂)_(m)C(O)—, and m may be an integer between 2 and 4,more preferably 2 or 3.

Particularly preferably, X may be a succinyl group with m being 2.

In another aspect, the present invention relates to a stabilized vitaminC derivative having a peptide molecule represented by the followingChemical Formula 2, or a pharmaceutically acceptable salt thereof:

wherein

R4 and R5 are —OC(O)(CH₂)pCH₃, or

and are different from each other;

X is —OC(O)(CH₂)mC(O)—;

is a natural or non-natural peptide molecule in which identical ordifferent amino acid residues are linked through amide bonds, and theamino acid residues are selected from the group consisting of glycine,lysine, histidine, serine, proline, hydroxyproline, threonine, glutamicacid, methionine, glutamine, and arginine;

R is a side chain of the amino acid;

n is an integer between 3 and 10;

m is an integer between 2 and 5;

p is an integer between 10 and 20;

W is

or glucose; and

R3 is selected from the group consisting of —N(CH₃)₂, —N(CH₂CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂C₆H₅, and —OH.

In a specific embodiment of the present invention, the compoundrepresented by Chemical Formula 2 may be a compound represented by thefollowing Chemical Formula 2a, 2b, 2c, or 2d:

In a preferred embodiment of the present invention,

in Chemical Formula 2, 2a, 2b, 2c, or 2d may be a peptide whereinidentical or different amino acid residues selected from the groupconsisting of glycine, lysine, histidine, serine, proline,hydroxyproline, threonine, arginine, glutamine, methionine, and glutamicacid are linked through amide bonding;

R is a side chain amino acid residue; and

n is an integer between 3 and 7.

More preferably, the peptide

may be a tri-, tetra-, penta-, or hepta-peptide, with n being an integerbetween 3 and 6, particularly preferably one selected from the groupconsisting of glycine-lysine-histidine, glycine-histidine-lysine,glycine-proline-hydroxyproline,lysine-threonine-threonine-lysine-serine, and glutamic acid-glutamicacid-methionine-glutamine-arginine-arginine.

X in Chemical Formula 2 may be —OC(O)(CH₂)_(m)C(O)—, and m may be aninteger between 2 and 4, more preferably 2 or 3.

Particularly preferably, X may be a succinyl group with m being 2.

In addition, —OC(O)(CH₂)_(p)CH₃ may be a fatty acid with p being aninteger between 10 and 20, more preferably an integer between 12 and 16.Particularly preferably, it may be a palmitate with p being 14.

In a preferred embodiments of the present invention, the stabilizedvitamin C derivative represented by Chemical Formula 1 or 2 may be oneselected from the group consisting of2-tetramethylphosphorodiamidic-5-(succinyklysine-threonine-threonine-lysine-serine)ascorbicacid,2-tetramethylphosphorodiamidic-6-(succinyl-lysine-threonine-threonine-lysine-serine)ascorbicacid,2-phospho-5-(succinyl-lysine-threonine-threonine-lysine-serine)ascorbicacid,2-phospho-6-(succinyl-lysine-threonine-threonine-lysine-serine)ascorbicacid,2-tetramethylphosphorodiamidic-5-(succinyl-glycine-lysine-histidine)ascorbicacid,2-tetramethylphosphorodiamidic-6-(succinyl-glycine-lysine-histidine)ascorbicacid, 2-phospho-5-(succinyl-glycine-lysine-histidine)ascorbic acid,2-phospho-6-(succinyl-glycine-lysine-histidine)ascorbic acid,2-tetramethylphosphorodiamidic-5-(succinyl-glycine-histidine-lysine)ascorbicacid,2-tetramethylphosphorodiamidic-6-(succinyl-glycine-histidine-lysine)ascorbicacid, 2-phospho-5-(succinyl-glycine-histidine-lysine)ascorbic acid,2-phospho-6-(succinyl-glycine-histidine-lysine)ascorbic acid,2-tetramethylphosphorodiamidic-5-(succinyl-glycine-proline-hydroxyproline)ascorbicacid,2-tetramethylphosphorodiamidic-6-(succinyl-glycine-proline-hydroxyproline)ascorbicacid, 2-phospho-5-(succinyl-glycine-proline-hydroxyproline)ascorbicacid, 2-phospho-6-(succinyl-glycine-proline-hydroxyproline)ascorbicacid, 2-tetramethylphosphorodiamidic-5-(succinyl-glutamic acid-glutamicacid-methionine-glutamine-arginine-arginine)ascorbic acid,2-tetramethylphosphorodiamidic-6-(succinyl-glutamic acid-glutamicacid-methionine-glutamine-arginine-arginine)ascorbic acid,2-phospho-5-(succinyl-glutamic acid-glutamicacid-methionine-glutamine-arginine-arginine)ascorbic acid,2-phospho-6-(succinyl-glutamic acid-glutamicacid-methionine-glutamine-arginine-arginine)ascorbic acid,2-tetramethylphosphorodiamidic-5-(succinyl-lysine-threonine-threonine-lysine-serine)-6-palmithylascorbic acid,2-tetramethylphosphorodiamidic-5-palmithyl-6-(succinyklysine-threonine-threonine-lysine-serine)ascorbicacid,2-phospho-5-(succinyl-lysine-threonine-threonine-lysine-serine)-6-palmithylascorbic acid,2-phospho-5-palmithyl-6-(succinyl-lysine-threonine-threonine-lysine-serine)ascorbicacid,2-tetramethylphosphorodiamidic-5-(succinyl-glycine-lysine-histidine)-6-palmithylascorbic acid,2-tetramethylphosphorodiamidic-5-palmithyl-6-(succinyl-glycine-lysine-histidine)ascorbicacid, 2-phospho-5-(succinyl-glycine-lysine-histidine)-6-palmithylascorbic acid,2-phospho-5-palmithyl-6-(succinyl-glycine-lysine-histidine)ascorbicacid,2-tetramethylphosphorodiamidic-5-(succinyl-glycine-histidine-lysine)-6-palmithylascorbic acid,2-tetramethylphosphorodiamidic-5-palmithyl-6-(succinyl-glycine-histidine-lysine)ascorbicacid, 2-phospho-5-(succinyl-glycine-histidine-lysine)-6-palmithylascorbic acid,2-phospho-5-palmithyl-6-(succinyl-glycine-histidine-lysine)ascorbicacid,2-tetramethylphosphorodiamidic-5-(succinyl-glycine-proline-hydroxyproline)-6-palmithylascorbic acid,2-tetramethylphosphorodiamidic-5-palmithyl-6-(succinyl-glycine-proline-hydroxyproline)ascorbicacid, 2-phospho-5-(succinyl-glycine-proline-hydroxyproline)-6-palmithylascorbic acid,2-phospho-5-palmithyl-6-(succinyl-glycine-proline-hydroxyproline)ascorbicacid, 2-tetramethylphosphorodiamidic-5-(succinyl-glutamic acid-glutamicacid-methionine-glutamine-arginine-arginine)-6-palmithyl ascorbic acid,2-tetramethylphosphorodiamidic-5-palmithyl-6-(succinyl-glutamicacid-glutamic acid-methionine-glutamine-arginine-arginine)ascorbic acid,2-phospho-5-(succinyl-glutamic acid-glutamicacid-methionine-glutamine-arginine-arginine)-6-palmithyl ascorbic acid,2-phospho-5-palmithyl-6-(succinyl-glutamic acid-glutamicacid-methionine-glutamine-arginine-arginine)ascorbic acid, andpharmaceutically acceptable salts thereof.

In the most preferred embodiment of the present invention, thestabilized vitamin C derivative represented by Chemical Formula 1 or 2may be one selected from the group consisting of2-tetramethylphosphorodiamidic-5-(succinyl-lysine-threonine-threonine-lysine-serine)ascorbicacid,2-phospho-5-(succinyl-lysine-threonine-threonine-lysine-serine)ascorbicacid,2-tetramethylphosphorodiamidic-5-(succinyl-glycine-lysine-histidine)ascorbicacid, 2-phospho-5-(succinyl-glycine-lysine-histidine)ascorbic acid,2-tetramethylphosphorodiamidic-5-(succinyl-glycine-histidine-lysine)ascorbicacid, 2-phospho-5-(succinyl-glycine-histidine-lysine)ascorbic acid,2-tetramethylphosphorodiamidic-5-(succinyl-glycine-proline-hydroxyproline)ascorbicacid, 2-phospho-5-(succinyl-glycine-proline-hydroxyproline)ascorbicacid, 2-tetramethylphosphorodiamidic-5-(succinyl-glutamic acid-glutamicacid-methionine-glutamine-arginine-arginine)ascorbic acid,2-phospho-5-(succinyl-glutamic acid-glutamicacid-methionine-glutamine-arginine-arginine)ascorbic acid,2-tetramethylphosphorodiamidic-5-palmithyl-6-(succinyl-lysine-threonine-threonine-lysine-serine)ascorbicacid,2-phospho-5-palmithyl-6-(succinyklysine-threonine-threonine-lysine-serine)ascorbicacid,2-tetramethylphosphorodiamidic-5-palmithyl-6-(succinyl-glycine-lysine-histidine)ascorbicacid,2-phospho-5-palmithyl-6-(succinyl-glycine-lysine-histidine)ascorbicacid,2-tetramethylphosphorodiamidic-5-palmithyl-6-(succinyl-glycine-histidine-lysine)ascorbicacid,2-phospho-5-palmithyl-6-(succinyl-glycine-histidine-lysine)ascorbicacid,2-tetramethylphosphorodiamidic-5-palmithyl-6-(succinyl-glycine-proline-hydroxyproline)ascorbicacid,2-phospho-5-palmithyl-6-(succinyl-glycine-proline-hydroxyproline)ascorbicacid, 2-tetramethylphosphorodiamidic-5-palmithyl-6-(succinyl-glutamicacid-glutamic acid-methionine-glutamine-arginine-arginine)ascorbic acid,2-phospho-5-palmithyl-6-(succinyl-glutamic acid-glutamicacid-methionine-glutamine-arginine-arginine)ascorbic acid, andpharmaceutically acceptable salts thereof.

In this description, the “natural peptide” refers to a peptide havingα-amino acids selected from the group consisting of alanine, valine,leucine, isoleucine, proline, phenylalanine, tryptophan, methionine,glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine,lysine; arginine, histidine, aspartic acid, and glutamic acid. And, the“non-natural peptide” refers to a peptide having amino acids not encodedby a nucleic acid codon. It includes, for example, D-isomers of theabove natural α-amino acids; Aib (aminobutyric acid), bAib(3-aminoisobutyric acid), Nva (norvaline), β-Ala, Aad (2-aminoadipicacid), bAad (3-aminoadipic acid), Abu (2-aminobutyric acid), Gaba(γ-aminobutyric acid), Acp (6-aminocaproic acid), Dbu(2,4-diaminobutyric acid), α-aminopimelic acid, TMSA(trimethylsilyl-Ala), alle(allo-isoleucine), Nle (norleucine), tert-Leu,Cit (citrulline), Orn, Dpm (2,2′-diaminopimelic acid), Dpr(2,3-diaminopropionic acid), α- or β-Nal, Cha (cyclohexyl-Ala),hydroxyproline, Sar (sarcosine), etc.; cyclic amino acids; N-α-alkylatedamino acids, e.g., MeGly (N-α-methylglycine), EtGly (N-α-ethylglycine),and EtAsn(N-α-ethylasparagine); and amino acids with two side chainsubstituents at the α-carbon, etc.

As used herein, the term “peptide” refers to a polymer made up of 10 orless (preferably 3 to 10) amino acid residues linked through amide bonds(or peptide bonds). The peptide may be one obtained by treating aprotein extracted from the body with a protease, or one synthesized by agene recombination and protein expression system. Preferably, thepeptide may be synthesized in vitro using a peptide synthesizer, etc.The peptide may be a derivative with a particular atom or atom groupsubstituted by a hydroxyl group, etc. Hereinbelow, the COOH terminus ofa peptide is abbreviated as C-terminus, and the NH₂ terminus asN-terminus.

In the present invention, the peptide may particularly include acollagen-producing peptide. The “collagen-producing peptide” refers to afragment of a collagen protein, which can be utilized to promote thesynthesis of collagen or can be used as a constituent of collagen. Forexample, a tri-, tetra-, penta, or hepta-peptide selected from the groupconsisting glycine-lysine-histidine, glycine-histidine-lysine,glycine-proline-hydroxyproline,lysine-threonine-threonine-lysine-serine, and glutamic acid-glutamicacid-methionine-glutamine-arginine-arginine, which maybe used in anembodiment of the present invention, promotes the synthesis of collagenand glycosaminoglycan in the dermis, thereby increasing moistureretention ability and elasticity of the dermis and improving wrinkleproblems. Particularly, the pentapeptidelysine-threonine-threonine-lysine-serine is the C-terminal fragment ofcollagen type I, which is important with regard to skin connectivetissues, wrinkle formation, and moisturization. It is reported that thepeptide promotes the synthesis of fibronectin, which helps intercellularcommunication between collagen types I and III and between cells duringthe cell culturing of fibroblasts that produce collagen, and thesynthesis of the growth factor β-TGF (Kou Katayama et al., 1991,Biochemistry 30: 7097-7104) and promotes its transcription by binding tothe collagen gene promoter (Kou Katayama et al., 1993, The Journal ofBiological Chemistry 268(14): 9941-9944).

As used herein, the “pharmaceutically acceptable salt” includes saltsderived from a pharmaceutically acceptable inorganic acid, organic acid,or base. Examples of salts derived from pharmaceutically acceptableacids may be selected from the group consisting of hydrochloric acid,bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid,maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylicacid, succinic acid, toluene-p-sulfonic acid, tartaric acid, aceticacid, citric acid, methanesulfonic acid, formic acid, benzoic acid,malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc.Examples salts derived from of pharmaceutically acceptable bases arealkali metals such as sodium, etc., alkaline earth metals such asmagnesium, etc., ammonium, etc.

As used herein, “ascorbic acid” has the same meaning as “vitamin C.”

The vitamin C derivative of the present invention has been invented tosolve the low stability problem of conventional vitamin C, in spite ofsuperior antioxidant activity, skin whitening, and regeneration ability,etc. The stability is improved by introducing a phosphate derivative orglucose at the C-2 position of vitamin C, and is further improved byintroducing a particular peptide at the C-5 or C-6 position.Particularly, if the peptide is a collagen-producing peptide, a skincondition improvement effect, including wrinkle improvement, skinelasticity improvement, etc., can be attained additionally by collagen.

In still another aspect, the present invention provides a method ofpreparing a stabilized vitamin C derivative comprising the steps of:

introducing a phosphoryl group, a phosphate derivative or glucose at theC-2 position of vitamin C to substitute for hydroxyl group at C-2position of vitamin C; and

introducing -OH or a fatty acid ester having 12 to 22 carbon atoms atone of C-5 and C-6 positions, and introducing a peptide having 3 to 10amino acid residues at the other position.

In an embodiment of the present invention, the preparation method of astabilized vitamin C derivative of the present invention may be a methodof preparing the vitamin C derivative with a peptide moleculerepresented by Chemical Formula 1, or a pharmaceutically acceptable saltthereof, which comprises the steps of:

introducing glucose, a phosphoryl group or a phosphate derivativerepresented by the following Chemical Formula 6 to substitute for thehydroxyl group at the C-2 position of vitamin C (the step of introducinga benzyl or other protecting group to the hydroxyl group of the C-3position may be optionally included, if required);

generating a hydroxyl group at one of the C-5 and C-6 positions ofvitamin C and a dicarboxylic acid HOOC(CH₂)_(m)COOH (m is an integerbetween 2 and 5) at the other position through an ester bond; and

linking a peptide having 3 to 10 amino acid residues to the dicarboxylicacid through an amide bond.

In an embodiment of the present invention, the preparation method of thevitamin C derivative represented by Chemical Formula 1 may include, butnot be limited to, the steps of:

reacting the 5,6-isopropylidiene-ascorbic acid represented by thefollowing Chemical Formula 3 with a benzyl halide represented by thefollowing Chemical Formula 4 to introduce a benzyl group at the hydroxylgroup of the C-3 position of 5,6-isopropylidiene-ascorbic acid andobtain the benzyl-substituted 5,6-isopropylidiene-ascorbic acidrepresented by the following Chemical Formula 5;

reacting the ascorbic acid derivative represented by Chemical Formula 5with a phosphorodiamidic halide represented by the following ChemicalFormula 6 to introduce a phosphorodiamide group at the hydroxyl group ofthe C-2 position and obtain the benzyl- and phosphorodiamide-substituted5,6-isopropylidiene-ascorbic acid represented by the following ChemicalFormula 7;

opening the ring of the ascorbic acid derivative represented by ChemicalFormula 7, and generating a hydroxyl group at one of the C-5 and C-6positions and introducing a dicarboxylic acid represented by thefollowing Chemical Formula 8 to obtain the ascorbic acid derivativebonded with the dicarboxylic acid through ester bonding, which isrepresented by the following Chemical Formula 9;

linking the ascorbic acid derivative represented by Chemical Formula 9with a peptide represented by the following Chemical Formula 10 throughamide bonding; and removing the benzyl group:

wherein

m is an integer between 2 and 5;

Bn is benzyl;

Y is chlorine, bromine, fluorine, or iodine;

R3′ is dimethylamine, diethylamine, propylamine, or benzylamine;

R1′ and R2′ are —OH or —OC(O)(CH₂)mC(O)OH, and are different from eachother, wherein they are not simultaneously —OH or —OC(O)(CH₂)mC(O)OH;

R is a protected or unprotected side chain of a natural or non-naturalamino acid residue; and

n is an integer between 3 and 10.

In a more preferred embodiment, the vitamin C derivative represented byChemical Formula 1 and a pharmaceutically acceptable salt thereof may beprepared as follows.

First, a benzyl halide represented by Chemical Formula 4, preferablybenzyl chloride (Y=chlorine), is added to the5,6-isopropylidiene-ascorbic acid represented by Chemical Formula 3 andreaction is performed for 3 to 5 hours, preferably for 4 hours, at 4 to50° C., preferably at 20 to 30° C., particularly preferably at 25° C.,to introduce the benzyl group at the hydroxyl group of the C-3 positionof ascorbic acid (vitamin C). Subsequently, a phosphorodiamidic haliderepresented by Chemical Formula 6, preferablyN,N,N′,N′-tetramethylphosphorodiamidic chloride, is added to thebenzyl-substituted compound represented by Chemical Formula 5, in whichthe benzyl group is introduced at the hydroxyl group of the C-3position, and reaction is performed for 5 to 7 hours, preferably for 6hours, at 4 to 70° C., preferably at 20 to 30° C., particularlypreferably at 25° C., to obtain the compound represented by ChemicalFormula 7, in which the benzyl group is introduced at the hydroxyl groupof the C-3 position of ascorbic acid and theN,N,N′,N′-tetramethylphosphorodiamide group is introduced at the C-2position.

To the resultant compound, an acid, preferably trifluoroacetic acid, isadded and reaction is performed for 3 to 5 hours, preferably for 4hours, to open the 5,6-isopropylidiene ring. Subsequently, adicarboxylic acid represented by Chemical Formula 8, preferably succinicacid, is added and reaction is performed for 15 to 17 hours, preferablyfor 16 hours, at 4 to 70° C., preferably at 20 to 30° C., particularlypreferably at 25° C., with ascorbic acid to form the compoundrepresented by Chemical Formula 9 through ester bonding. Then, theprotected NH₂ group of the N-terminus is linked with the dicarboxylicacid through amide bonding by the conventional solid phase synthesis inresin, and the protecting group is removed (deprotection).

The reaction may be performed under an anhydrous condition, for exampleby using an anhydrous organic solvent. For the anhydrous organicsolvent, any commonly used anhydrous organic solvent, e.g.,dimethylformamide, dimethylsulfoxide, N-methylpyrrolidinone,dichloromethane, etc., preferably dimethylformamide, may be used. Inaddition, a condensing agent, for example DCC(N,N′-dicyclohexylcarbodiimide), HOBT (N-hydroxybenzotriazole), EDC[N-(3-dimethylaminopropyl)-N′-ethyl-carbodiimide], PyBOP(benzotriazole-1-yl-oxy-tris-pyrrolidinophosphoniumhexafluorophosphate), HBTU[2-(1H-benzotriazole-1-yl)-1,3,3-tetramethyluroniumhexafluorophosphate], etc. may be used in the reaction.

In another embodiment of the present invention, the preparation methodof a stabilized vitamin C derivative of the present invention may be apreparation method of the stabilized vitamin C derivative with a peptidemolecule represented by Chemical Formula 2 or a pharmaceuticallyacceptable salt thereof, which includes the steps of:

introducing glucose, a phosphoryl group, or a phosphate derivativerepresented by the following Chemical Formula 6 at the hydroxyl group ofthe C-2 position of vitamin C (the step of introducing a benzyl group orother protecting group to the hydroxyl group of the C-3 position may befurther added, if required); and

introducing a fatty acid ester having 12 to 22 carbon atoms to thehydroxyl group of any one of C-5 and C-6 positions of vitamin C andintroducing the dicarboxylic acid HOOC(CH₂)_(m)COOH (m is an integerbetween 2 and 5) at the other hydroxyl group through ester bonding andlinking with a peptide having 3 to 10 amino acid residues through amidebonding.

In an embodiment of the present invention, the preparation method of thevitamin C derivative represented by Chemical Formula 2 or apharmaceutically acceptable salt thereof comprises the steps of:

reacting the 5,6-isopropylidiene-ascorbic acid represented by ChemicalFormula 3 with a benzyl halide represented by the following ChemicalFormula 4 to introduce the benzyl group at the hydroxyl group of the C-3position of the 5,6-isopropylidiene-ascorbic acid and obtain the5,6-isopropylidiene-ascorbic acid represented by the following ChemicalFormula 5;

reacting the ascorbic acid derivative represented by Chemical Formula 5with a phosphorodiamidic halide represented by the following ChemicalFormula 6 to introduce the phosphorodiamide group at the hydroxyl groupof the C-2 position and obtain the 5,6-isopropylidiene-ascorbic acidrepresented by the following Chemical Formula 7, which is substitutedwith the benzyl group and the phosphorodiamide group;

opening the ring of the ascorbic acid derivative represented by ChemicalFormula 7, reacting the hydroxyl group of any one of the C-5 and C-6positions with a fatty acid represented by the following ChemicalFormula 11 and reacting the other hydroxyl group with dicarboxylic acidrepresented by the following Chemical Formula 8 to obtain the ascorbicacid derivative represented by the following Chemical Formula 12, whichis bound to the fatty acid and the dicarboxylic acid through esterbonding;

reacting the resultant ascorbic acid derivative represented by ChemicalFormula 9 with a peptide represented by the following Chemical Formula10 to form an amide bonding with the dicarboxylic acid; and removing thebenzyl group:

wherein

m is an integer between 2 and 5;

Bn is benzyl;

Y is chlorine, bromine, fluorine, or iodine;

R3′ is dimethylamine, diethylamine, propylamine, or benzylamine;

R4′

R5′ are —OC(O)(CH₂)pCH₃ or —OC(O)(CH₂)mC(O)OH, and are different fromeach other, wherein they are not simultaneously —OC(O)(CH₂)pCH₃ or—OC(O)(CH₂)mC(O)OH;

R is a protected or unprotected side chain of a natural or non-naturalamino acid residue;

n is an integer between 3 and 10; and

p is an integer between 10 and 20.

In a more preferred embodiment, the vitamin C derivative represented byChemical Formula 2 or a pharmaceutically acceptable salt thereof may beprepared as follows.

First, a benzyl halide represented by Chemical Formula 4, preferablybenzyl chloride (Y=chlorine), is added to the ascorbic acid derivativerepresented by Chemical Formula 3 and reaction is performed for 3 to 5hours, preferably for 4 hours, at 4 to 70° C., preferably at 20 to 30°C., particularly preferably at 25° C. to obtain the ascorbic acidderivative represented by Chemical Formula 5, in which the hydroxylgroup of the C-3 position of ascorbic acid is substituted by the benzylgroup. A phosphorodiamidic halide, preferablyN,N,N′,N′-tetramethylphosphorodiamidic chloride, is added to theresultant ascorbic acid derivative and reaction is performed for 5 to 7hours, preferably for 6 hours, at 4 to 70° C., preferably at 20 to 30°C., particularly preferably at 25° C., to obtain the ascorbic acidderivative represented by Chemical Formula 7, in which the benzyl groupis introduced at the hydroxyl group of the C-3 position of ascorbic acidand the N,N,N′,N′-tetramethylphosphorodiamide is introduced at thehydroxyl group of the C-2 position. An acid, preferably trifluoroaceticacid, is added to the resultant ascorbic acid derivative and reaction isperformed for 3 to 5 hours, preferably for 4 hours, to open the5,6-isopropylidiene ring. Then, a fatty acid represented by ChemicalFormula 11, preferably palmitic acid, is added and reaction is performedfor 15 to 17 hours, preferably for 16 hours, at 4 to 70° C., preferablyat 20 to 30° C., particularly preferably at 25° C., to link with thehydroxyl group of the C-5 or C-6 position of the ring-opened ascorbicacid derivative through ester bonding. Subsequently, a dicarboxylic acidrepresented by Chemical Formula 8, preferably succinic acid, is addedand reaction is performed for 15 to 17 hours, preferably for 16 hours,at 4 to 70° C., preferably at 20 to 30° C., particularly preferably at25° C., to link the hydroxyl group of the C-5 or C-6 position of thering-opened ascorbic acid not bonded to the fatty acid through esterbonding to obtain the ascorbic acid derivative represented by ChemicalFormula 12. The protected NH₂ group of the N-terminus of the resultantascorbic acid derivative is linked with the dicarboxylic acid throughamide bonding by the conventional solid phase synthesis in resin anddeprotection is performed.

The reaction may be performed under an anhydrous condition, for example,using an anhydrous organic solvent. The anhydrous organic solvent may beany commonly used anhydrous organic solvent, e.g., dimethylformamide,dimethylsulfoxide, N-methylpyrrolidinone, dichloromethane, etc.,preferably dimethylformamide. In addition, a condensing agent, e.g., DCC(N,N′-dicyclohexylcarbodiimide), HOBT (N-hydroxybenzotriazole), EDC[N-(3-dimethylaminopropyl)-N′-ethyl-carbodiimide], PyBOP(benzotriazole-1-yl-oxy-tris-pyrrolidinophosphoniumhexafluorophosphate), HBTU[2-(1H-benzotriazole-1-yl)-1,3,3-tetramethyluroniumhexafluorophosphate], etc., may be used during the reaction.

In the present invention, any condensing agent, catalyst, solvent, etc.,commonly used in the related field may be used, so long as it does notnegatively affect the reaction.

The obtained stabilized vitamin C derivative with a peptide molecule ofthe present invention represented by Chemical Formula 1 or 2 may bepurified by the conventional separation and purification method, forexample by recrystallization or column chromatography.

In the preparation method of the compound represented by ChemicalFormula 1 or 2 in accordance with the present invention, the peptidethat is linked with the ascorbic acid whose hydroxyl group is protectedwith the benzyl group may be prepared by solid phase synthesis. Forexample, after protecting each of the hydroxyl groups of ascorbic acid,a succinyl group is selectively introduced to the unprotected hydroxylgroup. Then, the succinyl-substituted ascorbic acid is reacted with apeptide prepared by solid phase synthesis in resin at the N-terminus ofthe peptide, and the protecting groups are removed from the ascorbicacid and the peptide.

The following schemes are examples of the preparation method of thevitamin C derivative compound represented by Chemical Formula 1 or 2 insolid phase or solution phase. Scheme 1 is a process for preparing thecompounds represented by Chemical Formulae 1a and 1c, Scheme 2 is aprocess for preparing the compounds represented by Chemical Formulae 1band 1d, Scheme 3 is a process for preparing the compounds represented byChemical Formulae 2b and 2d, and Scheme 4 is the process for preparingthe compounds represented by Chemical Formulae 2a and 2c.

In another aspect, the present invention provides an acyltion agentincluding the compound represented by the following Chemical Formula 9or 12, or a method of acylating a peptide or protein using the same:

wherein

R3′ is dimethylamine, diethylamine, propylamine, or benzylamine;

R1′ and R2′ are —OH, or —OC(O)(CH₂)mC(O)OH, and are different from eachother, wherein they are not simultaneously —OH, or —OC(O)(CH₂)mC(O)OH;and

R4′

R5′ are —OC(O)(CH₂)pCH₃ or —OC(O)(CH₂)mC(O)OH, and are different fromeach other, wherein they are not simultaneously —OC(O)(CH₂)pCH₃ or—OC(O)(CH₂)mC(O)OH.

As described above, the compound represented by Chemical Formula 9 or 12may be used to acylate a compound having a functional group that can besubstituted by the acyl group. For example, the compound represented byChemical Formula 9 or 12 may be reacted with a compound having an aminogroup to form amide bonds. The compound having an amino group may be apeptide, as defined in this description, or a protein.

The stabilized vitamin C derivative with a peptide molecule of thepresent invention or a pharmaceutically acceptable salt thereof hassuperior antioxidation, antiaging, skin wrinkle improvement, and skinwhitening effects without skin irritation, skin sensitization,phototoxicity, etc. in addition, since the vitamin C derivative of thepresent invention has lipophilicity similar to that of skin, it hassuperior skin permeability. When a collagen-producing peptide is bound,the vitamin C derivative may have a better skin wrinkle improvementeffect. Further, since the vitamin C derivative of the present inventionis bound to a phosphate derivative, it has significantly improvedstability.

Thus, in another aspect, the present invention provides a compositionfor skin improvement including at least one selected from the groupconsisting of the compound represented by Chemical Formula 1, thecompound represented by Chemical Formula 2, and pharmaceuticallyacceptable salts thereof as active ingredient. Preferably, thecomposition is used for skin application for transdermal administration.Because of the skin permeability and stability of the active ingredient,the composition is superior in offering the effects of skin anti-aging,wrinkle improvement, skin roughness improvement, skin whitening, skinmoisturization, etc., and is thereby effective in improving skinconditions and preventing aggravation of skin conditions.

In this description, “for skin application” means that the compositionis superior in offering the effects of skin moisturizing, skinwhitening, wrinkle improvement, skin roughness improvement, promotion oftransdermal absorption, etc., and thus, is used to improve skinconditions or to prevent aggravation of skin conditions. The preparationfor skin application of the present invention may be used for cosmetics,medicine, and particularly for medicine for skin application such asointments, etc. Preferably, it is used for cosmetics, without anyparticular limitation in the form.

In the composition in accordance with the present invention, the contentof the active ingredient, or the at least one substance selected fromthe group consisting of the compound represented by Chemical Formula 1,the compound represented by Chemical Formula 2, and pharmaceuticallyacceptable salts thereof, can be determined adequately, considering theuse, form, purpose, etc., of the composition. A content of at least0.001% is preferable, because a substantial skin improvement effectcannot be expected with a content smaller than 0.001%. In a preferredembodiment of the present invention, the composition for skinimprovement of the present invention may comprise the active ingredientat 0.001 to 50% (w/w), preferably at 0.001 to 5% (w/w), and morepreferably at 0.01 to 3% (w/w).

In addition, the composition for skin improvement of the presentinvention may include a commonly used solvent, for example at least oneselected from the group consisting of ethanol, glycerin, butyleneglycol, propylene glycol, Glycereth-26, Methylgluceth-20, isocetylmyristate, isocetyl octanoate, octyld:odecyl myristate, octyldodecanol,isostearyl isostearate, cetyl octanoate, and neopentyl glycol dicaprate.When the composition of the present invention is prepared using thesesolvents, the solubility of the compound varies slightly, depending onthe particular compound and the mixing ratio of the solvents. However,those skilled in the art can select the particular solvent to be usedand its content adequately, depending on the characteristics of theproduct. Further, the composition for skin improvement of the presentinvention may include an adequate additive such as perfume, pigment,antiseptic, vehicle, etc., depending on the form of the composition.

The composition for skin improvement of the present invention may beprepared into a variety of product forms, including skin ointment, skinsoftener, nourishing lotion, nourishing cream, massage cream, essence,pack, etc. For example, when the composition is used for a skinointment, it may include, in addition to the active ingredient, or thecompound represented by Chemical Formula 1 or 2, 50.0-97.0 wt % ofvaseline and 0.1-5.0 wt % of polyoxyethylene oleyl ether phosphate. Whenused for a skin softener, the composition may comprise 1.0-10.0 wt % ofpolyalcohol, such as propylene glycol, glycerin, etc., and 0.05-2.0 wt %of surfactant, such as polyethylene oleyl ether, polyoxyethylenehydrogenated castor oil, etc. Further, when used for a nourishing lotionor a nourishing cream, it may include, in addition to the activeingredient, 5.0-20.0 wt % of oil, such as squalene, Vaseline, andoctyldodecanol, and 3.0-15.0 wt % of ethanol, stearyl alcohol, beeswax,etc. When used for an essence, it may include 5.0-30.0 wt % ofpolyalcohol, such as glycerin, propylene glycol, etc. When used for amassage cream, the composition may include, in addition to the activeingredient, 30.0-70.0 wt % of an oil, such as liquid paraffin, vaseline,isononly isononanoate, etc. And, when used for a pack, it may beprepared into a peel-off pack including 5.0-20.0 wt % of polyvinylalcohol or a wash-off pack including general-purpose emulsion cosmeticsand 5.0-30.0 wt % of pigment, such as kaolin, talc, zinc oxide, titaniumdioxide, etc.

Further, the composition for skin improvement of the present inventionmay include substances commonly used in general cosmetics, for exampleoil, water, a surfactant, a moisturizer, a low alcohol, a thickener, achelating agent, a pigment, an antiseptic, a perfume, etc., as required.

In other aspect, the present invention provides a method of stabilizingvitamin C. The method may include the steps of:

introducing glucose or a phosphoryl group or a phosphate derivativerepresented by Chemical Formula 6 to substitute for a hydroxyl group ata C-2 position of vitamin C;

binding a peptide molecule having 3 to 10 amino acid residues selectedfrom the group consisting of glycine, lysine, histidine, serine,proline, hydroxyproline, threonine, glutamic acid, methionine,glutamine, and arginine, to a C-5 or C-6 position of vitamin C through adicarboxylic acid represented by the following Chemical Formula 8.

The peptide molecule may be selected from glycine-histidine-lysine,glycine-lysine-histidine, glycine-proline-hydroxyproline,lysine-threonine-threonine-lysine-serine, and glutamic acid-glutamicacid-methionine-glutamine-arginine-arginine.

The present invention is further explained in more detail with referenceto the following examples. These examples, however, should not beinterpreted as limiting the scope of the present invention in anymanner.

Examples Example 1 Synthesis of3-O-benzyl-2-O-tetramethylphosphorodiamidic-6-O-triphenylmethyl-5-O-succinyl-ascorbicAcid

3-O-benzyl-2-O-tetramethylphosphorodiamidic-6-O-triphenylmethyl-5-O-succinyl-ascorbicacid was prepared by the following scheme.

(a) Protection with Benzyl Group

5,6-Isopropylidiene-L-ascorbic acid (290 g, 1.34 mol) was dissolved in 2L of dimethylformamide, and solid potassium carbonate (92.7 g, 0.67 mol)was added thereto. Benzyl chloride (115 g, 0.67 mmol) was added to thesolution and stirring was performed at 25° C. for 4 hours.Dimethylformamide was removed from the solution under reduced pressure.The resultant solution was dissolved in 2 L of ethyl acetate and 2 L ofwater was added. The organic layer was extracted using a separatoryfunnel. This procedure was repeated 3 times to remove water-solubleimpurities. 500 g of anhydrous sodium sulfate was added to the extractedorganic layer to remove water. The remaining solution was concentratedunder reduced pressure to obtain 300 g of 5,6-isopropylidiene--ascorbicacid in which the hydroxyl group at the C-3 position is protected by abenzyl group.

(b) Introduction of Phosphate Derivative

300 g of the obtained compound and 120 g of dimethylaminopyridine weredissolved in 2 L of dichloromethane. Then, 180 g oftetramethylphosphorodiamidic chloride was added, and stirring wasperformed at 25° C. for 6 hours. Dichloromethane was removed from thesolution under reduced pressure. The resultant solution was dissolved in2 L of ethyl acetate and 2 L Of a 5% sodium hydrogen sulphate solutionwas added. The organic layer was extracted using a separatory funnel.500 g of anhydrous sodium sulfate was added to the extracted organiclayer to remove water. The remaining solution was concentrated underreduced pressure to obtain 300 g of 5,6-isopropylidiene-L-ascorbic acidin which tetramethylphosphorodiamide was introduced at the C-2 position.

(c) Ring Opening by Addition of Acid

300 g of the obtained compound was dissolved in 800 mL ofdichloromethane and 200 mL of 20% (v/v) trifluoroacetic acid (TFA) wasadded. Stirring was performed at 25° C. for 4 hours. Dichloromethane andTFA were removed from the solution under reduced pressure andrecrystallization was performed to obtain 250 g of a pale yellow,semi-solid, ring-opened ascorbic acid derivative.

(d) Protection of Hydroxyl Group at C-6 Position

250 g of the obtained compound and 64 g of triethylamine were dissolvedin 1 L of dichloromethane and 180 g of triphenylmethyl chloride wasadded. Stirring was performed at 25° C. for 24 hours. Dichloromethanewas removed from the solution under reduced pressure. The resultantsolution was dissolved in 2 L of ethyl acetate and 2 L of a 5% sodiumhydrogen sulphate solution was added. The organic layer was extractedusing a separatory funnel. 500 g of anhydrous sodium sulfate was addedto the extracted organic layer to remove water. The remaining solutionwas concentrated under reduced pressure to obtain 240 g of an ascorbicacid derivative in which the hydroxyl group at the C-6 position wasprotected by triphenylmethyl chloride.

(e) Synthesis of Ascorbic Acid Derivative in which Succinic Acid isIntroduced at the C-5 Position

240 g of the obtained compound was dissolved in 2 L of dichloromethaneand dimethylaminopyridine (1.1 eq.) and succinic acid (1.2 eq.) wereadded. Stirring was performed at 25° C. for 16 hours. Dichloromethanewas removed from the solution under reduced pressure. The resultantsolution was dissolved in 2 L of ethyl acetate and 2 L of a 5% sodiumhydrogen sulphate solution was added. The organic layer was extractedusing a separatory funnel. 500 g of anhydrous sodium sulfate was addedto the extracted organic layer to remove water. After filtration, theremaining solution was concentrated under reduced pressure. 270 g of apale yellow, semi-solid compound was obtained from the concentratedsolution by silica gel column chromatography (total yield: 27.1%). NMRanalysis results for the obtained compound,3-O-benzyl-2-O-tetramethylphosphorodiamidic-6-O-triphenylmethyl-5-O-succinyl-ascorbicacid are as follows.

¹H NMR (CDCl₃): 2.63 (tt, 4H, CH₂CH ₂COOH), 2.7 (m, 12H, P(O)(N(CH₃)₂)₂), 3.33 (tt, 2H, C-6-H ₂), 4.98 (s, 1H, C-5-H), 5.43 (t, H, C-4-H₂), 5.53 (dd, 2H, O—CH ₂-Ph), 7.33 (m, 20H, Ar—H)

Example 2 Synthesis of3-O-benzyl-2-O-tetramethylphosphorodiamidic-5-O-tert-butyloxycarbonyl-6-O-succinyl-ascorbicAcid

3-O-benzyl-2-O-tetramethylphosphorodiamidic-5-O-tert-butyloxycarbonyl-6-O-succinyl-ascorbicacid was prepared by the following scheme.

(a) Protection of Hydroxyl Group at the C-5 Position

3-O-benzyl-2-O-tetramethylphosphorodiamidic-6-O-triphenylmethyl-ascorbicacid (100 g, 0.1556 mol) and triethylamine (18 g) were dissolved in 1 Lof dichloromethane. Di-tent-butyl dicarbamate (37 g) was added to thesolution and stirring was performed at 25° C. for 12 hours.Dichloromethane was removed from the solution under reduced pressure.The resultant solution was dissolved in 1 L of ethyl acetate and 1 L ofa 5% sodium hydrogen sulphate solution was added. The organic layer wasextracted using a separatory funnel. This procedure was repeated 3 timesto remove water-soluble impurities. 300 g of anhydrous sodium sulfatewas added to the extracted organic layer to remove water. The remainingsolution was concentrated under reduced pressure to obtain 104 g of3-O-benzyl-2-O-tetramethylphosphorodlamidic-5-O-tert-butyloxycarbonyl-6-O-triphenylmethyl-ascorbicacid in which the hydroxyl group at the C-5 position was protected by atert-butyloxycarbonyl group.

(b) Deprotection of the C-6 Position

104 g of the obtained compound was dissolved in 1 L. of methyl alcoholand the cation exchange resin Dowex50w-8x was added. Stirring wasperformed at 60° C. for 24 hours. After filtration, methyl alcohol wasremoved under reduced pressure. The resultant solution was dissolved in1 L of ethyl acetate and 1 L of a 5% sodium bicarbonate solution wasadded. The organic layer was extracted using a separatory funnel. 300 gof anhydrous sodium sulfate was added to the extract to remove water.After filtration, the remaining solution was concentrated under reducedpressure to obtain 63 g of an ascorbic acid derivative in which the C-6position was deprotected and that had a hydroxyl group.

(c) Synthesis of Ascorbic Acid Derivative in which Succinic Acid isBound at the C-6 Position

63 g of the obtained compound was dissolved in 1 L of dichloromethaneand dimethylaminopyridine (1.1 eq.) and succinic acid (1.2 eq.) wereadded. Stirring was performed at 25° C. for 16 hours. Dichloromethanewas removed from the solution under reduced pressure. The resultantsolution was dissolved in 1 L of ethyl acetate and 1 L of a 5% sodiumhydrogen sulphate solution was added. The organic layer was extractedusing a separatory funnel. 300 g of anhydrous sodium sulfate was addedto the extract to remove water. After filtration, the remaining solutionwas concentrated under reduced pressure. 68 g of a pale yellow,semi-solid compound was obtained from the concentrated solution bysilica gel column chromatography (total yield: 25%). NMR analysisresults for the obtained compound,3-O-benzyl-2-O-tetramethylphosphorodiamidic-5-O-tert-butyloxycarbonyl-6-O-succinyl-ascorbicacid, are as follows.

¹H NMR (CDCl₃): 1.42 (s, 9H, OC(CH ₃)₃), 2.63 (tt, 4H, CH ₂CH ₂COOH),2.7 (m, 12H, P(O)(N(CH ₃)₂)₂), 3.33 (tt, 2H, C-6-H ₂), 4.98 (s, 1H,C-5-H), 5.43 (t, H, C-4-H ₂), 5.53 (dd, 2H, O—CH ₂-Ph), 7.33 (m, 5H,Ar—H)

Example 3 Synthesis of3-O-benzyl-2-O-tetramethylphosphorodiamidic-5-O-palmitoyl-6-O-succinyl-ascorbicAcid

3-O-benzyl-2-O-tetramethylphosphorodiamidic-5-O-palmitoyl-6-O-succinyl-ascorbicAcid was Prepared by the Following Scheme

(a) Introduction of Palmitoyl Group at the C-5 Position

10 g of3-O-benzyl-2-O-tetramethylphosphorodiamidic-6-O-triphenylmethyl-ascorbicacid and 2 g of triethylamine were dissolved in 100 mL ofdichloromethane and 5 g of palmitoyl chloride was added. Then, stirringwas performed at 25° C. for 16 hours. Dichloromethane was removed fromthe solution under reduced pressure. The resultant solution wasdissolved in 100 mL of ethyl acetate and 100 mL of a 5% sodium hydrogensulphate solution was added. The organic layer was extracted using aseparatory funnel. 30 g of anhydrous sodium sulfate was added to theextract to remove water. After filtration, the remaining solution wasconcentrated under reduced pressure to obtain 10 g of an ascorbic acidderivative in which a palmitoyl group is introduced at the C-5 position.

(b) Deprotection of the C-6 Position

10 g of the obtained compound was dissolved in 100 mL of methyl alcoholand the cation exchange resin Dowex50w-8x was added. Then, stirring wasperformed at 60° C. for 24 hours. After filtration, methyl alcohol wasremoved under reduced pressure. The resultant solution was dissolved in100 mL of ethyl acetate and 100 mL of a 5% sodium bicarbonate solutionwas added. The organic layer was extracted using a separatory funnel. 30g of anhydrous sodium sulfate was added to the extract to remove water.After filtration, the remaining solution was concentrated under reducedpressure to obtain 5 g of an ascorbic acid derivative in which the C-6position was deprotected and that had a hydroxyl group.

(c) Introduction of Succinic acid at the C-6 Position

5 g of the obtained compound was dissolved in 50 mL of dichloromethaneand dimethylaminopyridine (1.1 eq.) and succinic acid (1.2 eq.) wereadded. Then, stirring was performed at 25° C. for 16 hours.Dichloromethane was removed from the solution under reduced pressure.The resultant solution was dissolved in 50 mL of ethyl acetate and 50 mLof a 5% sodium hydrogen sulphate solution was added. The organic layerwas extracted using a separatory funnel. 10 g of anhydrous sodiumsulfate was added to the extract to remove water. After filtration, theremaining solution was concentrated under reduced pressure. 5 g of apale yellow, semi-solid compound was obtained from the concentratedsolution by silica gel column chromatography (total yield: 13.3%). NMRanalysis results for the obtained compound,3-O-benzyl-2-O-tetramethylphosphorodiamidic-5-O-palmitoyl-6-O-succinyl-ascorbicacid, are as follows.

¹H NMR (CDCl₃): 0.88 (t, 3H, C(O)CH₂(CH₂)₁₃CH ₃), 1.28 (m, 26H,C(O)CH₂(CH ₂)₁₃CH₃), 2.53 (t, 2H, C(O)CH ₂(CH₂ ₁₃CH₃), 2.59 (tt, 4H, CH₂CH ₂COOH), 2.7 (m, 12H, P(O)(N(CH ₃)₂)₂), 3.33 (tt, 2H, C-6-H ₂), 4.98(s, 1H, C-5-H), 5.43 (t, H, C-4-H ₂), 5.53 (dd, 2H, O—CH ₂—Ph), 7.33 (m,5H, Ar—H)

Example 4 Synthesis of3-O-benzyl-2-O-tetramethylphosphorodiamidic-6-O-palmitoyl-5-O-succinyl-ascorbicAcid

3-O-benzyl-2-O-tetramethylphosphorodiamidic-6-O-palmitoyl-5-O-succinyl-ascorbicacid was prepared by the following scheme.

(a) Protection of Hydroxyl Group at the C-5 Position

3-O-benzyl-2-O-tetramethylphosphorodiamidic-6-O-triphenylmethyl-ascorbicacid (10 g, 15,56 mmol) and triethylamine (1.8 g) were dissolved in 100mL of dichloromethane and di-tert-butyl dicarbamate (3.7 g) was added.Then, stirring was performed at 25° C. for 12 hours. Dichloromethane wasremoved from the solution under reduced pressure. The resultant solutionwas dissolved in 100 mL of ethyl acetate and 100 mL of a 5% sodiumhydrogen sulphate solution was added. The organic layer was extractedusing a separatory funnel. This procedure was repeated 3 times to removewater-soluble impurities. 30 g of anhydrous sodium sulfate was added tothe organic layer to remove water. After filtration, the remainingsolution was concentrated under reduced pressure to obtain 10 g of3-O-benzyl-2-O-tetramethylphosphorodiamidic-5-O-tert-butyloxycarbonyl-6-O-triphenylmethyl-ascorbicacid in which the hydroxyl group at the C-5 position is protected by atert-butyloxycarbonyl group.

(b) Deprotection of the C-6 Position

10 g of the obtained compound was dissolved in 100 mL of methyl alcoholand the cation exchange resin Dowex50w-8x was added. Then, stirring wasperformed at 60° C. for 24 hours. After filtration, methyl alcohol wasremoved under reduced pressure. The resultant solution was dissolved in100 mL of ethyl acetate and 100 mL of a 5% sodium bicarbonate solutionwas added. The organic layer was extracted using a separatory funnel. 30g of anhydrous sodium sulfate was added to the extract to remove water.After filtration, the remaining solution was concentrated under reducedpressure to obtain 6.3 g of an ascorbic acid derivative in which the C-6position was deprotected and that had a hydroxyl group.

(c) Introduction of Palmitoyl Group at the C-6 Position

6.3 g of the obtained compound and 2 g of triethylamine were dissolvedin 100 mL of dichloromethane and 5 g of palmitoyl chloride was added.Then, stirring was performed at 25° C. for 16 hours. Dichloromethane wasremoved from the solution under reduced pressure. The resultant solutionwas dissolved in 100 mL of ethyl acetate and 100 mL of a 5% sodiumhydrogen sulphate solution was added. The organic layer was extractedusing a separatory funnel. 30 g of anhydrous sodium sulfate was added tothe extract to remove water. After filtration, the remaining solutionwas concentrated under reduced pressure to obtain 10 g of an ascorbicacid derivative in which a palmitoyl was introduced at the C-6 position.

(d) Deprotection of the C-5 Position

10 g of the obtained compound was dissolved in 100 mL of methyl alcoholand the cation exchange resin Dowex50w-8x was added. Then, stirring wasperformed at 60° C. for 48 hours. After filtration, methyl alcohol wasremoved under reduced pressure. 100 mL of ethyl acetate was dissolved inthe resultant solution and 100 mL of a 5% sodium bicarbonate solutionwas added. The organic layer was extracted using a separatory funnel. 30g of anhydrous sodium sulfate was added to the extract to remove water.After filtration, the remaining solution was concentrated under reducedpressure to obtain 5 g of an ascorbic acid derivative in which the C-5position is deprotected and has a hydroxyl group.

(e) Introduction of Succinic acid at the C-5 Position

5 g of the obtained compound was dissolved in 50 mL of dichloromethaneand dimethylaminopyridine (1.1 eq.) and succinic acid (1.2 eq.) wereadded. Then, stirring was performed at 25° C. for 16 hours.Dichloromethane was removed from the solution under reduced pressure.The resultant solution was dissolved in 50 mL of ethyl acetate and 50 mLof a 5% sodium hydrogen sulphate solution was added. The organic layerwas extracted using a separatory funnel. 10 g of anhydrous sodiumsulfate was added to the extract to remove water. After filtration, theremaining solution was concentrated under reduced pressure. 4.8 g of apale yellow, semi-solid compound was obtained from the concentratedsolution by silica gel column chromatography (total yield: 12%). NMRanalysis results for the obtained compound,3-O-benzyl-2-O-tetramethylphosphorodiamidic-6-O-palmitoyl-5-O-succinyl-ascorbicacid, are as follows.

¹H NMR (CDCl₃): 0.88 (t, 3H, C(O)CH₂(CH₂)₁₃CH ₃), 1.28 (m, 26H,C(O)CH₂(CH ₂)₁₃CH₃), 2.53 (t, 2H, C(O)CH ₂(CH₂)₁₃CH₃), 2.59 (tt, 4H, CH₂CH ₂COOH), 2.7 (m, 12H, P(O)(N(CH ₃)₂)₂), 3.33 (tt, 2H, C-6-H ₂), 4.98(s, 1H, C-5-H), 5.43 (t, H, C-4-H ₂), 5.53 (dd, 2H, O—CH ₂—Ph), 7.33 (m,5H, Ar—H)

Example 5 Synthesis of Peptides

Peptide synthesis was performed by solid phase synthesis using Fmoc(9-fluorenylmethoxycarbonyl) as the protecting group of the N-α-aminoacid. The peptide chain was elongated by the HOBt-DCC(N-hydroxybenzotriazole-dicyclohexylcarodiimide) method (Wang C. Chan,Perter D. White, “Fmoc solid phase peptide synthesis” Oxford).Glycine-histidine-lysine (GHK), glycine-lysine-histidine (GKH),glycine-proline-hydroxyproline (GPO),lysine-threonine-threonine-lysine-serine (KTTKS), and glutamicacid-glutamic acid-methionine-glutamine-arginine-arginine (EEMQRR)peptides were synthesized.

Example 6 Synthesis of Stabilized Vitamin C Derivative with a PeptideMolecule 6-1: Synthesis of the Compound Represented by Chemical Formula1a

50 mL of a 20% piperidinelN-methylpyrrolidone solution was added to thepeptide prepared in Example 5 (1 mmol), in which up to the amino acid atthe N-terminus is coupled, to remove the Fmoc group. After washing withN-methylpyrrolidone and dichloromethane, the vitamin C derivativeprepared in Example 1 was coupled. After coupling was completed, thepeptide was washed several times with N-methylpyrrolidone anddichloromethane and dried with nitrogen gas. Then, 50 mL oftrifluoroacetic acid was added and reaction was performed at 25° C. for3 hours to remove the peptide protecting group and the triphenylmethylgroup that protects protects the alcohol group at the C-6 position ofvitamin C. The stabilized vitamin C derivative with a peptide moleculewas separated from the resin and the peptide was precipitated withdiethyl ether.

In order to remove the benzyl group that protects the alcohol group atthe C-3 position of vitamin C, 0.1 g of 10% Pd/C was added to 50 mL ofmethanol and stirring was performed at 25° C. for about 1 hour under ahydrogen atmosphere. Pd/C was removed using celite and the remainingsolution was concentrated under reduced pressure. The obtained vitamin Cderivative was purified by reverse phase high-performance liquid columnchromatography (Zobax, C8 300 Å, 21.1 mm×25 cm) using acetonitrilecontaining 0.1% trifluoroacetic acid and celite to obtain 0.5 g of thestabilized vitamin C derivative with a peptide molecule represented byChemical Formula 1a (compounds Ia-1 to Ia-5 in Table 1 below).

6-2: Synthesis of the Compound Represented by Chemical Formula 1b

50 mL of 20% piperidine/N-methylpyrrolidone solution was added to thepeptide prepared in Example 5 (1 mmol), in which up to the amino acid atthe N-terminus was coupled, to remove the Fmoc group. After washing withN-methylpyrrolidone and dichloromethane, the vitamin C derivativeprepared in Example 2 was coupled. After coupling was completed, thepeptide was washed several times with N-methylpyrrolidone anddichloromethane and dried with nitrogen gas. Then, 50 mL oftrifluoroacetic acid was added and reaction was performed at 25° C. for3 hours to remove the peptide protecting group and thetert-butyloxycarbonyl group that protects the alcohol group at the C-5position of vitamin C. The stabilized vitamin C derivative with apeptide molecule was separated from the resin and the peptide wasprecipitated with diethyl ether.

In order to remove the benzyl group that protects the alcohol group atthe C-3 position of vitamin C, 0.1 g of 10% Pd/C was added to 50 mL ofmethanol and stirring was performed at 25° C. for about 1 hour under ahydrogen atmosphere. Pd/C was removed using celite and the remainingsolution was concentrated under reduced pressure. The obtained vitamin Cderivative was purified by reverse phase high-performance liquid columnchromatography (Zobax, C8 300 Å, 21.1 mm×25 cm) using acetonitrilecontaining 0.1% trifluoroacetic acid and celite to obtain 0.5 g of thestabilized vitamin C derivative with a peptide molecule represented byChemical Formula 1b (compounds Ib-1 to Ib-5 in Table 1).

6-3: Synthesis of the Compound Represented by Chemical Formula 1c

0.5 g of the compound represented by Chemical Formula la, which wasprepared in Example 6-1, was dissolved in 50 mL of third purified waterand 0.5 g of the cation exchange resin Dowex 50w-8x was added. Then,stirring was performed at 25° C. for 24 hours. After filtration, 500 mLof ethanol was added to the remaining solution to form solids, whichwere filtered and dried under vacuum to obtain 0.4 g of the stabilizedvitamin C derivative with a peptide molecule represented by ChemicalFormula //lc (compounds Ic-1 to Ic-5 in Table 1).

6-4: Synthesis of the Compound Represented by Chemical Formula 1d

0.5 g of the compound represented by Chemical Formula 1 b, which wasprepared in Example 6-2, was dissolved in 50 mL of third purified waterand 0.5 g of the cation exchange resin Dowex 50w-8x was added. Then,stirring was performed at 25° C. for 24 hours. After filtration, 500 mLof ethanol was added to the remaining solution to form solids, whichwere filtered and dried under vacuum to obtain 0.4 g of the stabilizedvitamin C derivative with a peptide molecule represented by ChemicalFormula 1d (compounds Id-1 to Id-5 in Table 1).

6-5: Synthesis of the Compound Represented by Chemical Formula 2a

50 mL of 20% piperidine/N-methylpyrrolidone solution was added to thepeptide prepared in Example 5 (1 mmol), in which up to the amino acid atthe N-terminus was coupled, to remove the Fmoc group. After washing withN-methylpyrrolidone and dichloromethane, the vitamin C derivativeprepared in Example 3 was coupled. After coupling was completed, thepeptide was washed several times with N-methylpyrrolidone anddichloromethane and dried with nitrogen gas. Then, 50 mL oftrifluoroacetic acid was added and reaction was performed at 25° C. for3 hours to remove the peptide protecting group. The stabilized vitamin Cderivative with a peptide molecule was separated from the resin and thepeptide was precipitated with diethyl ether.

In order to remove the benzyl group that protects the alcohol group atthe C-3 position of vitamin C, 0.1 g of 10% Pd/C was added to 50 mL ofmethanol and stirring was performed at 25° C. for about 1 hour under ahydrogen atmosphere. Pd/C was removed using celite and the remainingsolution was concentrated under reduced pressure. The obtained vitamin Cderivative was purified by reverse phase high-performance liquid columnchromatography (Zobax, C8 300 Å, 21.1 mm×25 cm) using acetonitrilecontaining 0.1% trifluoroacetic acid and celite to obtain 0.6 g of thestabilized vitamin C derivative with a peptide molecule represented byChemical Formula 2a (compounds IIa-1 to IIa-5 in Table 1).

6-6: Synthesis of the Compound Represented by Chemical Formula 2b

50 mL of 20% piperidine/N-methylpyrrolidone solution was added to thepeptide prepared in Example 5 (1 mmol), in which up to the amino acid atthe N-terminus was coupled, to remove the Fmoc group. After washing withN-methylpyrrolidone and dichloromethane, the vitamin C derivativeprepared in Example 4 was coupled. After coupling was completed, thepeptide was washed several times with N-methylpyrrolidone anddichloromethane and dried with nitrogen gas. Then, 50 mL oftrifluoroacetic acid was added and reaction was performed at 25° C. for3 hours to remove the peptide protecting group. The stabilized vitamin Cderivative with a peptide molecule was separated from the resin and thepeptide was precipitated with diethyl ether.

In order to remove the benzyl group that protects the alcohol group atthe C-3 position of vitamin C, 0.1 g of 10% Pd/C was added to 50 mL ofmethanol and stirring was performed at 25° C. for about 1 hour under ahydrogen atmosphere. Pd/C was removed using celite and the remainingsolution was concentrated under reduced pressure. The obtained vitamin Cderivative was purified by reverse phase high-performance liquid columnchromatography (Zobax, C8 300 A, 21.1 mm X 25 cm) using acetonitrilecontaining 0.1% trifluoroacetic acid and celite to obtain 0.6 g of thestabilized vitamin C derivative with a peptide molecule represented byChemical Formula 2b (compounds IIb-1 to IIb-5 in Table 1).

6-7: Synthesis of the Compound Represented by Chemical Formula 2c

0.5 g of the compound represented by Chemical Formula 2a, which wasprepared in Example 6-5, was dissolved in 50 mL of third purified waterand 0.5 g of the cation exchange resin Dowex 50w-8x was added. Then,stirring was performed at 25° C. for 24 hours. After filtration, 500 mLof ethanol was added to the remaining solution to form solids, whichwere filtered and dried under vacuum to obtain 0.5 g of the stabilizedvitamin C derivative with a peptide molecule represented by ChemicalFormula 2c (compounds IIc-1 to IIc-5 in Table 1).

6-8: Synthesis of the Compound Represented by Chemical Formula 2d

0.5 g of the compound represented by Chemical Formula 1b, which wasprepared in Example 6-2, was dissolved in 50 mL of third purified waterand 0.5 g of the cation exchange resin Dowex 50w-8x was added. Then,stirring was performed at 25° C. for 24 hours. After filtration, 500 mLof ethanol was added to the remaining solution to form solids, whichwere filtered and dried under vacuum to obtain 0.5 g of the stabilizedvitamin C derivative with a peptide molecule represented by ChemicalFormula 2d (compounds IId-1 to IId-5 in Table 1).

TABLE 1 MS molecular Compd. 5′ carbon of 6′ carbon of weight No. vitaminC vitamin C W Theoretical Measured Ia-1 -X-KTTKS —OH —OP(O)N(CH₃)

956.02 957.1 Ia-2 -X-GHK —OH —OP(O)N(CH₃)

732.72 733.4 Ia-3 -X-GKH —OH —OP(O)N(CH₃)

732.72 733.4 Ia-4 -X-GPO —OH —OP(O)N(CH₃)

677.62 678.1 Ia-5 -X-EEMQRR —OH —OP(O)N(CH₃)

1270.22 1271.1 Ib-1 —OH -X-KTTKS —OP(O)N(CH₃)

956.02 957.1 Ib-2 —OH -X-GHK —OP(O)N(CH₃)

732.72 733.4 Ib-3 —OH -X-GKH —OP(O)N(CH₃)

732.72 733.4 Ib-4 —OH -X-GPO —OP(O)N(CH₃)

677.62 678.1 Ib-5 —OH -X-EEMQRR —OP(O)N(CH₃)

1270.22 1271.1 Ic-1 -X-KTTKS —OH —OP(O)(CH)

901.88 902.2 Ic-2 -X-GHK —OH —OP(O)(CH)

678.58 679.3 Ic-3 -X-GKH —OH —OP(O)(CH)

678.58 679.3 Ic-4 -X-GPO —OH —OP(O)(CH)

623.48 624.2 Ic-5 -X-EEMQRR —OH —OP(O)(CH)

1186.08 1187.1 Id-1 —OH -X-KTTKS —OP(O)(CH)

901.88 902.2 Id-2 —OH -X-GHK —OP(O)(CH)

678.58 679.3 Id-3 —OH -X-GKH —OP(O)(CH)

678.58 679.3 Id-4 —OH -X-GPO —OP(O)(CH)

623.48 624.2 Id-4 —OH -X-EEMQRR —OP(O)(CH)

1186.08 1187.1 IIa-1 -X-KTTKS —OC(O)(CH

)

CH₃ —OP(O)N(CH₃)

1284.56 1285.3 IIa-2 -X-GHK —OC(O)(CH

)

CH₃ —OP(O)N(CH₃)

1061.26 1062.1 IIa-3 -X-GKH —OC(O)(CH

)

CH₃ —OP(O)N(CH₃)

1061.26 1062.1 IIa-4 -X-GPO —OC(O)(CH

)

CH₃ —OP(O)N(CH₃)

1006.16 1007.1 IIa-5 -X-EEMQRR —OC(O)(CH

)

CH₃ —OP(O)N(CH₃)

1568.76 1569.5 IIb-1 —OC(O)(CH

)

CH₃ -X-KTTKS —OP(O)N(CH₃)

1284.56 1286.3 IIb-2 —OC(O)(CH

)

CH₃ -X-GHK —OP(O)N(CH₃)

1061.26 1062.1 IIb-3 —OC(O)(CH

)

CH₃ -X-GKH —OP(O)N(CH₃)

1061.26 1062.1 IIb-4 —OC(O)(CH

)

CH₃ -X-GPO —OP(O)N(CH₃)

1006.16 1007.1 IIb-5 —OC(O)(CH

)

CH₃ -X-EEMQRR —OP(O)N(CH₃)

1568.76 1569.5 IIc-1 -X-KTTKS —OC(O)(CH

)

CH₃ —OP(O)(CH)

1230.42 1231.3 IIc-2 -X-GHK —OC(O)(CH

)

CH₃ —OP(O)(CH)

1007.12 1008.1 IIc-3 -X-GKH —OC(O)(CH

)

CH₃ —OP(O)(CH)

1007.12 1008.1 IIc-4 -X-GPO —OC(O)(CH

)

CH₃ —OP(O)(CH)

952.02 953.1 IIc-5 -X-EEMQRR —OC(O)(CH

)

CH₃ —OP(O)(CH)

1514.62 1515.4 IId-1 —OC(O)(CH

)

CH₃ -X-KTTKS —OP(O)(CH)

1230.42 1231.3 IId-2 —OC(O)(CH

)

CH₃ -X-GHK —OP(O)(CH)

1007.12 1008.1 IId-3 —OC(O)(CH

)

CH₃ -X-GKH —OP(O)(CH)

1007.12 1008.1 IId-4 —OC(O)(CH

)

CH₃ -X-GPO —OP(O)(CH)

952.02 953.1 IId-5 —OC(O)(CH

)

CH₃ -X-EEMQRR —OP(O)(CH)

1514.62 1515.4

indicates data missing or illegible when filed

In Table 1, the compounds Ia, Ib, Ic, Id, IIa, IIb, IIc, and IId are thecompounds represented by Chemical Formulae 1a, 1b, 1c, 1d, 2a, 2b, 2c,and 2d, respectively. [Compound Ia-1] ¹H NMR (D₂O): 0.95 (m, peptide-H),1.22 (m, peptide-H), 1.55 (m, peptide-H), 2.52 (m, 4H, CH ₂CH ₂CONH),2.70 (m, 12H, P(O)(N(CH ₃)₂)₂), 3.33 (tt, 2H, C-6-H ₂), 4.21 (m,peptide-H), 4.98 (s, 1H, C-5-H), 5.43 (t, H, C-4-H ₂)

[Compound Ia-2] ¹H NMR (D₂O): 0.93 (m, peptide-H), 1.22 (m, peptide-H),1.66 (m, peptide-H), 2.52 (m, 4H, CH ₂CH ₂CONH), 2.70 (m, 12H, P(O)(N(CH₃)₂)₂), 3.17 (m, peptide-H), 3.33 (tt, 2H, C-6-H ₂), 4.08 (s, 2H,peptide-H), 4.55 (m, peptide-H), 4.98 (s, 1H, C-5-H), 5.43 (t, H, C-4-H₂)

[Compound Ia-3] ¹H NMR (D₂O): 0.93 (m, peptide-H), 1.22 (m, peptide-H),1.66 (m, peptide-H), 2.52 (m, 4H, CH ₂CH ₂CONH), 2.70 (m, 12H, P(O)(N(CH₃)₂)₂), 3.17 (m, peptide-H), 3.33 (tt, 2H, C-6-H ₂), 4.08 (s, 2H,peptide-H), 4.55 (m, peptide-H), 4.98 (s, 1H, C-5-H), 5.43 (t, H, C-4-H)

[Compound Ia-4] ¹H NMR (D₂O): 2.01 (m, peptide-H), 2.52 (m, 4H, CH ₂CH₂CONH), 2.70 (m, 12H, P(O)(N(CH ₃)₂)₂), 3.33 (tt, 2H, C-6-H ₂), 3.51 (m,peptide-H), 4.09 (s, 2H, peptide-H), 4.40 (m, peptide-H), 4.98 (s, 1H,C-5-H), 5.43 (t, H, C-4-H ₂)

[Compound la-5] ¹H NMR (D₂O): 0.91 (m, peptide-H), 1.55 (m, peptide-H),2.13 (m, peptide-H), 2.42 (m, peptide-H), 2.52 (m, 4H, CH ₂CH ₂CONH),2.61 (m, peptide-H), 2.70 (m, 12H, P(O)(N(CH ₃)₂)₂), 3.33 (tt, 2H,C-6-H2), 4.53 (m, peptide-H), 4.98 (s, 1H, C-5-H), 5.43 (t; H, C-4-H2)

[Compound Ib-1] ¹H NMR (D₂O): 0.95 (m, peptide-H), 1.22 (m, peptide-H),1.55 (m, peptide-H), 2.52 (m, 4H, CH ₂CH ₂CONH), 2.70 (m, 12H, P(O)(N(CH₃)₂)₂), 3.33 (tt, 2H, C-6-H ₂), 4.21 (m, peptide-H), 4.98 (s, 1H,C-5-H), 5.43 (t, H, C-4-H ₂)

[Compound Ib-2] ¹H NMR (D₂O): 0.93 (m, peptide-H), 1.22 (m, peptide-H),1.66 (m, peptide-H), 2.52 (m, 4H, CH ₂CH ₂CONH), 2.70 (m, 12H, P(O)(N(CH₃)₂)₂), 3.17 (m, peptide-H), 3.33 (tt, 2H, C-6-H ₂), 4.08 (s, 2H,peptide-H), 4.55 (m, peptide-H), 4.98 (s, 1H, C-5-H), 5.43 (t, H, C-4-H₂) [Compound Ib-3] ¹H NMR (D₂O): 0.93 (m, peptide-H), 1.22 (m,peptide-H), 1.66 (m, peptide-H), 2.52 (m, 4H, CH ₂CH ₂CONH), 2.70 (m,12H, P(O)(N(CH ₃)₂)₂), 3.17 (m, peptide-H), 3.33 (tt, 2H, C-6-H ₂), 4.08(s, 2H, peptide-H), 4.55 (m, peptide-H), 4.98 (s, 1H, C-5-H), 5.43 (t,H, C-4-H ₂)

[Compound Ib-4] ¹H NMR (D₂O): 2.01 (m, peptide-H), 2.52 (m, 4H, CH ₂CH₂CONH), 2.70 (m, 12H, P(O)(N(CH ₃)₂)₂), 3.33 (tt, 2H, C-6-H ₂), 3.51 (m,peptide-H), 4.09 (s, 2H, peptide-H), 4.40 (m, peptide-H), 4.98 (s, 1H,C-5-H), 5.43 (t, H, C-4-H ₂)

[Compound Ib-5] ¹H NMR (D₂O): 0.91 (m, peptide-H), 1.55 (m, peptide-H),2.13 (m, peptide-H), 2.42 (m, peptide-H), 2.52 (m, 4H, CH ₂CH ₂CONH),2.61 (m, peptide-H), 2.70 (m, 12H, P(O)(N(CH ₃)₂)₂), 3.33 (tt, 2H, C-6-H₂), 4.53 (m, peptide-H), 4.98 (s, 1H, C-5-H), 5.43 (t, H, C-4-H ₂)compound Ic-1] ¹H NMR (D₂O): 0.95 (m, peptide-H), 1.22 (m, peptide-H),1.55 (m, peptide-H), 2.52 (m, 4H, CH ₂CH ₂CONH), 3.33 (tt, 2H, C-6-H ₂),4.21 (m, peptide-H), 4.98 (s, 1H, C-5-H), 5.43 (t, H, C-4-H ₂)

[Compound Ic-2] ¹H NMR (D₂O): 0.93 (m, peptide-H), 1.22 (m, peptide-H),1.66 (m, peptide-H), 2.52 (m, 4H, CH ₂CH2CONH), 3.17 (m, peptide-H),3.33 (tt, 2H, C-6-H ₂), 4.08 (s, 2H, peptide-H), 4.55 (m, peptide-H),4.98 (s, 1H, C-5-H), 5.43 (t, H, C-4-H ₂)

[Compound Ic-3] ¹H NMR (D₂O): 0.93 (m, peptide-H), 1.22 (m, peptide-H),1.66 (m, peptide-H), 2.52 (m, 4H, CH ₂CH ₂CONH), 3.17 (m, peptide-H),3.33 (tt, 2H, C-6-H ₂), 4.08 (s, 2H, peptide-H), 4.55 (m, peptide-H),4.98 (s, 1H, C-5-H), 5.43 (t, H, C-4-H ₂)

[Compound Ic-4] ¹H NMR (D₂O): 2.01 (m, peptide-H), 2.52 (m, 4H, CH ₂CH₂CONH), 3.33 (tt, 2H, C-6-H ₂), 3.51 (m, peptide-H), 4.09 (s, 2H,peptide-H), 4.40 (m, peptide-H), 4.98 (s, 1H, C-5-H), 5.43 (t, H, C-4-H₂)

[Compound Ic-5] ¹H NMR (D₂O): 0.91 (m, peptide-H), 1.55 (m, peptide-H),2.13 (m, peptide-H), 2.42 (m, peptide-H), 2.52 (m, 4H, CH ₂CH ₂CONH),2.61 (m, peptide-H), 3.33 (tt, 2H, C-6-H ₂), 4.53 (m, peptide-H), 4.98(s, 1H, C-5-H), 5.43 (t, H, C-4-H ₂)

[Compound Id-1] ¹H NMR (D₂O): 0.95 (m, peptide-H), 1.22 (m, peptide-H),1.55 (m, peptide-H), 2.52 (m, 4H, CH ₂CH ₂CONH), 3.33 (tt, 2H, C-6-H ₂),4.21 (m, peptide-H), 4.98 (s, 1H, C-5-H), 5.43 (t, H, C-4-H ₂)

[Compound Id-2] ¹H NMR (D₂O): 0.93 (m, peptide-H), 1.22 (m, peptide-H),1.66 (m, peptide-H), 2.52 (m, 4H, CH ₂CH ₂CONH), 3.17 (m, peptide-H),3.33 (tt, 2H, C-6-H ₂), 4.08 (s, 2H, peptide-H), 4.55 (m, peptide-H),4.98 (s, 1H, C-5-H), 5.43 (t, H, C-4-H ₂)

[Compound Id-3] ¹H NMR (D₂O): 0.93 (m, peptide-H), 1.22 (m, peptide-H),1.66 (m, peptide-H), 2.52 (m, 4H, CH ₂CH ₂CONH), 3.17 (m, peptide-H),3.33 (tt, 2H, C-6-H ₂), 4.08 (s, 2H, peptide-H), 4.55 (m, peptide-H),4.98 (s, 1H, C-5-H), 5.43 (t, H, C-H ₂)

[Compound Id-4] ¹H NMR (D₂O): 2.01 (m, peptide-H), 2.52 (m, 4H, CH ₂CH₂CONH), 3.33 (tt, 2H, C-6-H ₂), 3.51 (m, peptide-H), 4.09 (s, 2H,peptide-H), 4.40 (m, peptide-H), 4.98 (s, 1H, C-5-H), 5.43 (t, H, C-4-H₂)

[Compound Id-5] ¹H NMR (D₂O): 0.91 (m, peptide-H), 1.55 (m, peptide-H),2.13 (m, peptide-H), 2.42 (m, peptide-H), 2.52 (m, 4H, CH ₂CH ₂CONH),2.61 (m, peptide-H), 3.33 (tt, 2H, C-6-H ₂), 4.53 (m, peptide-H), 4.98(s, 1H, C-5-H), 5.43 (t, H, C-4-H ₂)

[Compound IIa-1] ¹H NMR (D₂O): 0.88 (t, 3H, C(O)CH₂(CH₂)₁₃CH ₃), 0.95(m, peptide-H), 1.22 (m, peptide-H), 1.28 (m, 26H, C(O)CH₂(CH ₂)13CH₃),1.55 (m, peptide-H), 2.52 (m, 4H, CH ₂CH ₂CONH), 2.53 (t, 2H, C(O)CH₂(CH₂)₁₃CH₃), 2.70 (m, 12H, P(O)(N(CH3)2)2), 3.33 (tt, 2H, C-6-H ₂),4.21 (m, peptide-H), 4.98 (s, 1H, C-5-H), 5.43 (t, H, C-4-H ₂)

[Compound IIa-2] ¹H NMR (D₂O): 0.88 (t, 3H, C(O)CH₂(CH₂)₁₃CH ₃), 0.93(m, peptide-H), 1.22 (m, peptide-H), 1.28 (m, 26H, C(O)CH₂(CH ₂)₁₃CH₃),1.66 (m, peptide-H), 2.52 (m, 4H, CH ₂CH ₂CONH), 2.53 (t, 2H, C(O)CH₂(CH₂)₁₃CH₃), 2.70 (m, 12H, P(O)(N(CH ₃)₂)₂), 3.17 (m, peptide-H), 3.33(tt, 2H, C-6-H ₂), 4.08 (s, 2H, peptide-H), 4.55 (m, peptide-H), 4.98(s, 1H, C-5-H), 5.43 (t, H, C-4-H ₂)

[Compound IIa-3] ¹H NMR (D₂O): 0.88 (t, 3H, C(O)CH₂(CH₂)₁₃CH ₃), 0.93(m, peptide-H), 1.22 (m, peptide-H), 1.28 (m, 26H, C(O)CH₂(CH ₂)₁₃CH₃),1.66 (m, peptide-H), 2.52 (m, 4H, CH ₂CH ₂CONH), 2.53 (t, 2H, C(O)CH₂(CH₂)₁₃CH₃), 2.70 (m, 12H, P(O)(N(CH ₃)₂)₂), 3.17 (m, peptide-H), 3.33(tt, 2H, C-6-H ₂), 4.08 (s, 2H, peptide-H), 4.55 (m, peptide-H), 4.98(s, 1H, C-5-H), 5.43 (t, H, C-4-H ₂)

[Compound IIa-4] ¹H NMR (D₂O): 0.88 (t, 3H, C(O)CH₂(CH₂)₁₃CH ₃), 1.28(m, 26H, C(O)CH₂(CH ₂)₁₃CH₃), 2.01 (m, peptide-H), 2.52 (m, 4H, CH ₂CH₂CONH), 2.53 (t, 2H, C(O)CH ₂(CH₂)₁₃CH₃), 2.70 (m, 12H, P(O)(N(CH₃)₂)₂), 3.33 (tt, 2H, C-6-H ₂), 3.51 (m, peptide-H), 4.09 (s, 2H,peptide-H), 4.40 (m, peptide-H), 4.98 (s, 1H, C-5-H), 5.43 (t, H, C-4-H₂)

[Compound IIa-5] ¹H NMR (D₂O): 0.88 (t, 3H, C(O)CH₂(CH₂)₁₃CH ₃), 0.91(m, peptide-H), 1.28 (m, 26H, C(O)CH₂(CH ₂)₁₃CH₃), 1.55 (m, peptide-H),2.13 (m, peptide-H), 2.42 (m, peptide-H), 2.52 (m, 4H, CH ₂CH ₂CONH),2.53 (t, 2H, C(O)CH ₂(CH₂)₁₃CH₃), 2.61 (m, peptide-H), 2.70 (m, 12H,P(O)(N(CH ₃)₂)₂), 3.33 (tt, 2H, C-6-H ₂), 4.53 (m, peptide-H), 4.98 (s,1H, C-5-H), 5.43 (t, H, C-4-H ₂)

[Compound IIb-1] ¹H NMR (D₂O): 0.88 (t, 3H, C(O)CH₂(CH₂)₁₃CH ₃), 0.95(m, peptide-H), 1.22 (m, peptide-H), 1.28 (m, 26H, C(O)CH₂(CH ₂)₁₃CH₃),1.55 (m, peptide-H), 2.52 (m, 4H, CH ₂CH ₂CONH), 2.53 (t, 2H, C(O)CH₂(CH₂)₁₃CH₃), 2.70 (m, 12H, P(O)(N(CH ₃)₂)₂), 3.33 (tt, 2H, C-6-H ₂),4.21 (m, peptide-H), 4.98 (s, 1H, C-5-H), 5.43 (t, H, C-4-H ₂)

[Compound IIb-2]¹H NMR (D₂O): 0.88 (t, 3H, C(O)CH₂(CH₂)₁₃CH ₃), 0.93 (m,peptide-H), 1.22 (m, peptide-H), 1.28 (m, 26H, C(O)CH₂(CH ₂)₁₃CH₃), 1.66(m, peptide-H), 2.52 (m, 4H, CH ₂CH ₂CONH), 2.53 (t, 2H, C(O)CH₂(CH₂)₁₃CH₃), 2.70 (m, 12H, P(O)(N(CH ₃)₂)₂), 3.17 (m, peptide-H), 3.33(tt, 2H, C-6-H ₂), 4.08 (s, 2H, peptide-H), 4.55 (m, peptide-H), 4.98(s, 1H, C-5-H), 5.43 (t, H, C-4-H ₂)

[Compound IIb-3] ¹H NMR (D₂O): 0.88 (t, 3H, C(O)CH₂(CH₂)₁₃CH ₃), 0.93(m, peptide-H), 1.22 (m, peptide-H), 1.28 (m, 26H, C(O)CH₂(CH ₂)₁₃CH₃),1.66 (m, peptide-H), 2.52 (m, 4H, CH ₂CH ₂CONH), 2.53 (t, 2H, C(O)CH₂(CH₂)₁₃CH₃), 2.70 (m, 12H, P(O)(N(CH ₃)₂)₂), 3.17 (m, peptide-H), 3.33(tt, 2H, C-6-H ₂), 4.08 (s, 2H, peptide-H), 4.55 (m, peptide-H), 4.98(s, 1H, C-5-H), 5.43 (t, H, C-4-H ₂)

[Compound IIb-4] ¹H NMR (D₂O): 0.88 (t, 3H, C(O)CH₂(CH₂)₁₃CH ₃), 1.28(m, 26H, C(O)CH₂(CH ₂)₁₃CH₃), 2.01 (m, peptide-H), 2.52 (m, 4H, CH ₂CH₂CONH), 2.53 (t, 2H, C(O)CH ₂(CH₂)₁₃CH₃), 2.70 (m, 12H, P(O)(N(CH₃)₂)₂), 3.33 (tt, 2H, C-6-H ₂), 3.51 (m, peptide-H), 4.09 peptide-H),4.40 (m, peptide-H), 4.98 (s, 1H, C-5-H), 5.43 (t, H, C-4-H ₂)

[Compound IIb-5] ¹H NMR (D₂O): 0.88 (t, 3H, C(O)CH₂(CH₂)₁₃CH ₃), 0.91(m, peptide-H), 1.28 (m, 26H, C(O)CH₂(CH ₂)₁₃CH₃), 1.55 (m, peptide-H),2.13 (m, peptide-H), 2.42 (m, peptide-H), 2.52 (m, 4H, CH ₂CH ₂CONH),2.53 (t, 2H, C(O)CH ₂(CH₂)₁₃CH₃), 2.61 (m, peptide-H), 2.70 (m, 12H,P(O)(N(CH ₃)₂)₂), 3.33 (tt, 2H, C-6-H ₂), 4.53 (m, peptide-H), 4.98 (s,1H, C-5-H), 5.43 (t, H, C-4-H ₂)

[Compound IIc-1] ¹H NMR (D₂O): 0.88 (t, 3H, C(O)CH₂(CH₂)₁₃CH ₃), 0.95(m, peptide-H), ;1.22 (m, peptide-H), 1.28 (m, 26H, C(O)CH₂(CH ₂)₁₃CH₃),1.55 ‘(m, peptide-H), 2.52 (m, 4H, CH ₂CH ₂CONH), 2.53 (t, 2H, C(O)CH₂(CH₂)₁₃CH₃), 3.33 (tt, 2H, C-6-H ₂), 4.21 (m, peptide-H), 4.98 (s, 1H,C-5-H), 5.43 (t, H, C-4-H ₂)

[Compound IIc-2] ¹H NMR (D₂O): 0.88 (t, 3H, C(O)CH₂(CH₂)₁₃CH ₃), 0.93(m, peptide-H), 1.22 (m, peptide-H), 1.28 (m, 26H, C(O)CH₂(CH ₂)₁₃CH₃),1.66 (m, peptide-H), 2.52 (m, 4H, CH ₂CH ₂CONH), 2.53 (t, 2H, C(O)CH₂(CH₂)₁₃CH₃), 3.17 (m, peptide-H), 3.33 (tt, 2H, C-6-H ₂), 4.08 (s, 2H,peptide-H), 4.55 (m, peptide-H), 4.98 (s, 1H, C-5-H), 5.43 (t, H, C-4-H₂)

[Compound IIc-3] ¹H NMR (D₂O): 0.88 (t, 3H, C(O)CH₂(CH₂)₁₃CH ₃), 0.93(m, peptide-H), 1.22 (m, peptide-H), 1.28 (m, 26H, C(O)CH₂(CH ₂)₁₃CH₃),1.66 (m, peptide-H), 2.52 (m, 4H, CH ₂CH ₂CONH), 2.53 (t, 2H, C(O)CH₂(CH₂)₁₃CH₃), 3.17 (m, peptide-H), 3.33 (tt, 2H, C-6-H ₂), 4.08 (s, 2H,peptide-H), 4.55 (m, peptide-H), 4.98 (s, 1H, C-5-H), 5.43 (t, H, C-4-H₂)

[Compound IIc-4] ¹H NMR (D₂O): 0.88 (t, 3H, C(O)CH₂(CH₂)₁₃CH ₃), 1.28(m, 26H, C(O)CH₂(CH ₂)₁₃CH₃), 2.01 (m, peptide-H), 2.52 (m, 4H, CH ₂CH₂CONH), 2.53 (t, 2H, C(O)CH ₂(CH₂)₁₃CH₃), 3.33 (tt, 2H, C-6-H ₂), 3.51(m, peptide-H), 4.09 (s, 2H, peptide-H), 4.40 (m, peptide-H), 4.98 (s,1H, C-5-H), 5.43 (t, H, C-4-H ₂)

[Compound IIc-5] ¹H NMR (D₂O): 0.88 (t, 3H, C(O)CH₂(CH₂)₁₃CH ₃), 0.91(m, peptide-H), 1.28 (m, 26H, C(O)CH₂(CH ₂)₁₃CH₃), 1.55 (m, peptide-H),2.13 (m, peptide-H), 2.42 (m, peptide-H), 2.52 (m, 4H, CH ₂CH ₂CONH),2.53 (t, 2H, C(O)CH ₂(CH₂)₁₃CH₃), 2.61 (m, peptide-H), 3.33 (tt, 2H,C-6-H ₂), 4.53 (m, peptide-H), 4.98 (s, 1H, C-5-H), 5.43 (t, H, C-4-H ₂)

[Compound IId-1] ¹H NMR (D₂O): 0.88 (t, 3H, C(O)CH₂(CH₂)₁₃CH ₃), 0.95(m, peptide-H), 1.22 (m, peptide-H), 1.28 (m, 26H, C(O)CH₂(CH ₂)₁₃CH₃),1.55 (m, peptide-H), 2.52 (m, 4H, CH ₂CH ₂CONH), 2.53 (t, 2H, C(O)CH₂(CH₂)₁₃CH₃), 3.33 (tt, 2H, C-6-H ₂), 4.21 (m, peptide-H), 4.98 (s, 1H,C-5-H), 5.43 (t, H, C-4-H ₂)

[Compound IId-2] ¹H NMR (D₂O): 0.88 (t, 3H, C(O)CH₂(CH₂)₁₃CH ₃), 0.93(m, peptide-H), 1.22 (m, peptide-H), 1.28 (m, 26H, C(O)CH₂(CH ₂)₁₃CH₃),1.66 (m, peptide-H), 2.52 (m, 4H, CH ₂CH ₂CONH), 2.53 (t, 2H, C(O)CH₂(CH₂)₁₃CH₃), 3.17 (m, peptide-H), 3.33 (tt, 2H, C-6-H ₂), 4.08 (s, 2H,peptide-H), 4.55 (m, peptide-H), 4.98 (s, 1H, C-5-H), 5.43 (t, H, C-4-H₂)

[Compound IId-3] ¹H NMR (D₂O): 0.88 (t, 3H, C(O)CH₂(CH₂)₁₃CH ₃), 0.93(m, peptide-H), 1.22 (m, peptide-H), 1.28 (m, 26H, C(O)CH₂(CH ₂)₁₃CH₃),1.66 (m, peptide-H), 2.52 (m, 4H, CH ₂CH ₂CONH), 2.53 (t, 2H, C(O)CH₂(CH₂)₁₃CH₃), 3.17 (m, peptide-H), 3.33 (tt, 2H, C-6-H ₂),

4.08 (s, 2H, peptide-H), 4.55 (m, peptide-H), 4.98 (s, 1H, C-5-H), 5.43(t, H, C-4-H ₂)

[Compound IId-4] ¹H NMR (D₂O): 0.88 (t, 3H, C(O)CH₂(CH₂)₁₃CH ₃), 1.28(m, 26H, C(O)CH₂(CH ₂)₁₃CH₃), 2.01 (m, peptide-H), 2.52 (m, 4H, CH ₂CH₂CONH), 2.53 (t, 2H, C(O)CH ₂(CH₂)₁₃CH₃), 3.33 (tt, 2H, C-6-H ₂), 3.51(m, peptide-H), 4.09 (s, 2H, peptide-H), 4.40 (m, peptide-H), 4.98 (s,1H, C-5-H), 5.43 (t, H, C-4-H ₂)

[Compound IId-5] ¹H NMR (D₂O): 0.88 (t, 3H, C(O)CH₂(CH₂)₁₃CH ₃), 0.91(m, peptide-H), 1.28 (m, 26H, C(O)CH₂(CH ₂)₁₃CH₃), 1.55 (m, peptide-H),2.13 (m, peptide-H), 2.42 (m, peptide-H), 2.52 (m, 4H, CH ₂CH ₂CONH),2.53 (t, 2H, C(O)CH ₂(CH₂)₁₃CH₃), 2.61 (m, peptide-H), 3.33 (tt, 2H,C-6-H ₂), 4.53 (m, peptide-H), 4.98 (s, 1H, C-5-H), 5.43 (t, H, C-4-H ₂)

Experimental Example 1 Collagen Biosynthesis

The active ingredient substance of the present invention was added tohuman skin fibroblasts to test the capability of promoting collagensynthesis at the cell level.

Biosynthesis of collagen was determined by the ELISA assay method. Theactive ingredient substance was not added to the control group. Therelative capability of promoting collagen synthesis was calculated, withthat of the control group being 100%. Details are as follows.

3000 human neonatal dermal fibroblasts (Cambrex) were grouped into a96-well plate for cell culture and were cultured for 24 hours in anincubator using DMEM (Dulbecco's Modified Eagle Media, Gibco BRL)containing 0.1% FBS (fetal bovine serum) under the condition of 37° C.and 5% CO₂. Each test substance was dissolved in DMEM containing 0.1%FBS to a concentration of 10 μM. After treating 200 μL of the solution,the cells were cultured for 72 hours. The supernatant was treated to a96-well plate in which the type 1 collagen antibody was coated. After 2hours of reaction at room temperature, the supematant was removed. Afterwashing with 0.05% Tween-20 PBS (PBST), a biotin-bound secondaryantibody was treated in the 96-well plate at room temperature. After 1hour of reaction, the remaining supernatant was removed. After washingwith PBST, SA-HRP (streptavidin-horseradish peroxidase, Sigma) was boundto measure the bound collagen. After treating with TMB (3,3′-5,5′tetramethylbenzidine, Sigma), reaction was performed at room temperaturefor 15 minutes, while blocking light. Reaction was stopped using 1 Nsulfuric acid and light absorbance was measured at 450 nm.

The results are shown in FIG. 1. As seen in FIG. 1, the compound Uc-1 ofthe present invention showed much superior capability of promotingcollagen synthesis than vitamin C (Vc), magnesium ascorbyl phosphate(MAP), succinoyl ascorbyl pentapeptide (Vc-KTTKS), and palmitoylpentapeptide (Pal-KTTKS). Also, the compounds Ic-2, Ic-3, Ic-4, and Ic-5of the present invention had superior capability in promoting collagensynthesis. Thus, it can be seen that the compounds Ic-1, Ic-2, Ic-3,Ic-4, and Ic-5 of the present invention have superior capability ofpromoting collagen synthesis, in addition to improved stability.

Experimental Example 2 Cell Toxicity

In order to prove the primary safety for use as cosmetics, cell toxicitywas measured for the compounds Ic-1, Is-2, Ic-3, Ic-4, and Ic-5 of thepresent invention, vitamin C (Vc), magnesium ascorbyl phosphate (MAP),succinoyl ascorbyl pentapeptide (Vc-KTTKS), and palmitoyl pentapeptide(Pal-KTTKS). 3000 human neonatal dermal fibroblasts (Cambrex) weregrouped into a 96-well plate for cell culture and were cultured for 24hours in an incubator using DMEM (Gibco BRL) at 0.1% under the conditionof 37° C. and 5% CO₂. Cell toxicity was measured 48 hours after treatingwith the test substance by the MTT assay (Mossman T., 1983, Journal ofImmunological Methods 65, 55-63).

The results are shown in FIG. 2. As seen in FIG. 2, the compound Ic-1 ofthe present invention did not show cell toxicity even at theconcentration of 5 mM, in contrast with vitamin C (Vc), succinoylascorbyl pentapeptide (Vc-KTTKS), and palmitoyl pentapeptide(Pal-KTTKS). In addition, the compounds Ic-2, Ic-3, Ic-4, and Ic-5 ofthe present invention did not show cell toxicity at 5 mM. Thus, thecompound of the present invention has superior safety.

Experimental Example 3 Stability

Each of the compounds Ia-1 and Ic-1 of the present invention, vitamin C,magnesium ascorbyl phosphate, and succinoyl ascorbyl pentapeptide weredissolved in third purified water to a concentration of 10 μg/mL.Stability of each substance was evaluated, while being maintained at 25°C. and 50° C. for 30 days, by observing the change of UV (254nm)absorbance. The obtained results are shown in Table 2.

TABLE 2 Remaining test substance (%) Magnesium Succinoyl Vitaminascorbyl ascorbyl Ia-1 Ic-1 C phosphate phosphate Days 25° c. 50° c. 25°c. 50° c. 25° c. 50° c. 25° c. 50° c. 25° c. 50° c. 0 100 100 100 100100 100 100 100 100 100 1 100 100 100 100 1 0 100 100 60 50 2 100 98 100100 0 0 100 100 20 10 3 100 97 100 100 0 0 100 100 10 0 5 100 95 100 990 0 100 99 0 0 10 99 90 100 99 0 0 100 99 0 0 20 98 85 100 98 0 0 100 980 0 30 97 80 100 98 0 0 100 98 0 0

As seen in Table 2, the compounds of the present invention had stabilitysuperior to that of vitamin C and succinoyl ascorbyl pentapeptide.Further, they showed comparable or better stability when compared withmagnesium ascorbyl phosphate, which is the most stable vitamin Cderivative known thus far. In addition, the compounds of the presentinvention showed superior capability of collagen biosynthesis.

As can be seen from the results of Test Examples 1 to 3, the vitamin Cderivative compound of the present invention shows comparable or betterstability, when compared with magnesium ascorbyl phosphate, which is themost stable vitamin C derivative known thus far, and has low celltoxicity and superior capability of collagen biosynthesis. Thus, it canbe used to improve skin conditions.

As is apparent from the above description, the stabilized vitamin Cderivative with a collagen-producing peptide in accordance with presentinvention has improved stability, safety, skin permeability, etc.,without toxicity such as skin irritation. With superior capability ofskin improvement and prevention of aggravation of skin conditions, itcan be utilized in such industrial fields as skin application medicines,cosmetics, etc.

1. A vitamin C derivative represented by the following Chemical Formula1, or a pharmaceutically acceptable salt thereof:

wherein R1 and R2 are —OH or

and are different from each other; X is —OC(O)(CH₂)mC(O)—;

is a peptide molecule wherein identical or different amino acid residuesare linked through amide bonds, and the amino acid residues are selectedfrom the group consisting of glycine, lysine, histidine, serine,proline, hydroxyproline, threonine, glutamic acid, methionine,glutamine, and arginine; R is a side chain of the amino acid; n is aninteger between 3 and 10; m is an inteaer between 2 and 5; W is

or glucose; and R3 is selected from the group consisting of —N(CH₃)₂,—N(CH₂CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂C₆H₅, and —OH.
 2. The vitamin Cderivative or a pharmaceutically acceptable salt thereof according toclaim 1, wherein R1 is

and R2 is —OH.
 3. The vitamin C derivative or a pharmaceuticallyacceptable salt thereof according to claim 1, wherein n is an integerbetween 3 and 6, and m is
 2. 4. The vitamin C derivative or apharmaceutically acceptable salt thereof according to claim 3, wherein

is a peptide molecule selected from glycine-histidine-lysine,glycine-lysine-histidine, glycine-proline-hydroxyproline,lysine-threonine-threonine-lysine-serine, and glutamic acid-glutamicacid-methionine-glutamine-arginine-arginine.
 5. The vitamin C derivativeor a pharmaceutically acceptable salt thereof according to claim 1,wherein R3 is a dimethylamine or hydroxyl group.
 6. The vitamin Cderivative or a pharmaceutically acceptable salt thereof according toclaim 1, wherein the vitamin C derivative is selected from the groupconsisting of2-tetramethylphosphorodiamidic-5-(succinyl-lysine-threonine-threonine-lysine-serine)ascorbicacid,2-phospho-5-(succinyl-lysine-threonine-threonine-lysine-serine)ascorbicacid,2-tetramethylphosphorodiamidic-5-(succinyl-glycine-lysine-histidine)ascorbicacid, 2-phospho-5-(succinyl-glycine-lysine-histidine)ascorbic acid,2-tetramethylphosphorodiamidic-5-(succinyl-glycine-histidine-lysine)ascorbicacid, 2-phospho-5-(succinyl-glycine-histidine-lysine)ascorbic acid,2-tetramethylphosphorodiamidic-5-(succinyl-glycine-proline-hydroxyproline)ascorbicacid, 2-phospho-5-(succinyl-glycine-proline-hydroxyproline)ascorbic acid2-tetramethylphosphorodiamidic-5-(succinyl-glutamic acid-glutamicacid-methionine-glutamine-arginine-arginine)ascorbic acid, and2-phospho-5-(succinyl-glutamic acid-glutamicacid-methionine-glutamine-arginine-arginine)ascorbic acid.
 7. A vitaminC derivative represented by the following Chemical Formula 2, or apharmaceutically acceptable salt thereof:

wherein R4 and R5 are —OC(O)(CH₂)pCH₃, or

and are different from each other; X is —OC(O)(CH₂)mC(O)—;

is a peptide molecule wherein identical or different amino acid residuesare linked through amide bonds, and the amino acid residues are selectedfrom the group consisting of glycine, lysine, histidine, serine,proline, hydroxyproline, threonine, glutamic acid, methionine,glutamine, and arginine; R is a side chain of the amino acid; n is aninteger between 3 and 10; m is an integer between 2 and 5; p is aninteger between 10 and 20; W is

or glucose; and R3 is selected from the group consisting of —N(CH₃)₂,—N(CH₂CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂C₆H₅, and —OH.
 8. The vitamin Cderivative or a pharmaceutically acceptable salt thereof according toclaim 7, wherein R4 is —OC(O)(CH₂)pCH₃, and R5 is


9. The vitamin C derivative or a pharmaceutically acceptable saltthereof according to claim 7, wherein n is an integer between 3 and 6,and p is
 14. 10. The vitamin C derivative or a pharmaceuticallyacceptable salt thereof according to claim 7, wherein

is a peptide molecule selected from the group consisting ofglycine-histidine-lysine, glycine-lysine-histidine,glycine-proline-hydroxyproline,lysine-threonine-threonine-lysine-serine, and glutamic acid-glutamicacid-methionine-glutamine-arginine-arginine.
 11. The vitamin Cderivative or a pharmaceutically acceptable salt thereof according toclaim 7, wherein R3 is dimethylamine or a hydroxyl group.
 12. Thevitamin C derivative or a pharmaceutically acceptable salt thereofaccording to claim 7, wherein the vitamin C derivative is selected fromthe group consisting of2-tetramethylphosphorodiamidic-5-palmithyl-6-(succinyl-lysine-threonine-threonine-lysine-serine)ascorbicacid,2-phospho-5-palmithyl-6-(succinyl-lysine-threonine-threonine-lysine-serine)ascorbicacid,2-tetramethylphosphorodiamidic-5-palmithyl-6-(succinyl-glycine-lysine-histidine)ascorbicacid,2-phospho-5-palmithyl-6-(succinyl-glycine-lysine-histidine)ascorbicacid,2-tetramethylphosphorodiamidic-5-palmithyl-6-(succinyl-glycine-histidine-lysine)ascorbicacid,2-phospho-5-palmithyl-6-(succinyl-glycine-histidine-lysine)ascorbicacid,2-tetramethylphosphorodiamidic-5-palmithyl-6-(succinyl-glycine-proline-hydroxyproline)ascorbicacid,2-phospho-5-palmithyl-6-(succinyl-glycine-proline-hydroxyproline)ascorbicacid2-tetramethylphosphorodiamidic-5-palmithyl-6-(succinyl-glutamicacid-glutamic acid-methionine-glutamine-arginine-arginine)ascorbic acid,and 2-phospho-5-palmithyl-6-(succinyl-glutamic acid-glutamicacid-methionine-glutamine-arginine-arginine)ascorbic acid.
 13. A methodof preparing the vitamin C derivative represented by Chemical Formula 1as defined in claim 1 comprising the steps of: introducing glucose or aphosphoryl group or a phosphate derivative represented by ChemicalFormula 6 to substitute for a hydroxyl group at the 2′ carbon positionof vitamin C; generating a hydroxyl group at any one of 5′ and 6′ carbonpositions of vitamin C, and a dicarboxylic acid represented by ChemicalFormula 8 at the other carbon position of vitamin C through an esterbond; and linking a peptide molecule having 3 to 10 amino acid residuesselected from the group consisting of glycine, lysine, histidine,serine, proline, hydroxyproline, threonine, glutamic acid, methionine,glutamine, and arginine, to the dicarboxylic acid through an amide bond:

wherein R3′ is selected from the group consisting of dimethylamine,diethylamine, propylamine, and benzylamine; Y is selected from the groupconsisting of chlorine, bromine, fluorine, and iodine; and m is aninteger between 2 and
 5. 14. The method according to claim 13, whereinthe number of the amino acid residues in the peptide molecule is 3 to 6,m is 2, and Y is chlorine.
 15. The method according to claim 13, whereinR3′ is dimethylamine.
 16. A method of preparing the vitamin C derivativerepresented by Chemical Formula 2 as defined in claim 7, comprising thesteps of: introducing glucose or a phosphoryl group or a phosphatederivative represented by Chemical Formula 6 to substitute for ahydroxyl group at the 2′ carbon position of vitamin C; binding a fattyacid represented by Chemical Formula 11 to a hydroxyl group present atany one of 5′ and 6′ carbon positions of vitamin C, and a dicarboxylicacid represented by Chemical Formula 8 to the other carbon position ofvitamin C through an ester bond; and linking a peptide molecule having 3to 10 amino acid residues selected from the group consisting of glycine,lysine, histidine, serine, proline, hydroxyproline, threonine, glutamicacid, methionine, glutamine, and arginine, to the dicarboxylic acidthrough an amide bond:

wherein R3′ is selected from the group consisting of dimethylamine,diethylamine, propylamine, and benzylamine; Y is selected from the groupconsisting of chlorine, bromine, fluorine, and iodine; m is an integerbetween 2 and 5; and p is an integer between 10 and
 20. 17. The methodaccording to claim 16, wherein the number of the amino acid residues inthe peptide molecule is 3 to 6, m is 2, and Y is chlorine.
 18. Themethod according to claim 16, wherein R3′ is dimethylamine.
 19. Anacylating agent of a peptide or a protein, containing a compoundrepresented by Chemical Formula 9 or 12:

wherein R3′ is selected from the group consisting of dimethylamine,diethylamine, propylamine, and benzylamine; R1′ and R2′ are —OH, or—OC(O)(CH2)mC(O)OH, and are different from each other, wherein they arenot simultaneously —OH, or —OC(O)(CH2)mC(O)OH; R4′ and R5′ are—OC(O)(CH₂)pCH₃ or —OC(O)(CH₂)mC(O)OH, and are different from eachother, wherein they are not simultaneously —OC(O)(CH₂)pCH₃ or—OC(O)(CH₂)mC(O)OH; m is an integer between 2 and 5; and p is an integerbetween 10 and
 20. 20. A method of acylating a peptide or a protein,using a compound represented by Chemical Formula 9 or 12:

wherein R3′ is selected from the group consisting of dimethylamine,diethylamine, propylamine, and benzylamine; R1′ and R2′ are —OH, or—OC(O)(CH2)mC(O)OH, and are different from each other, wherein they arenot simultaneously —OH or —OC(O)(CH2)mC(O)OH; R4′ and R5′ are—OC(O)(CH₂)pCH₃ or —OC(O)(CH₂)mC(O)OH, and are different from eachother, wherein they are not simultaneously —OC(O)(CH₂)pCH₃ or—OC(O)(CH₂)mC(O)OH; m is an integer between 2 and 5; and p is an integerbetween 10 and
 20. 21. A composition for preventing skin agingcontaining at least one selected from the vitamin C derivative asdefined in claim 1, and pharmaceutically acceptable salts thereof as anactive ingredient.
 22. A composition for improving skin wrinklingcontaining at least one selected from the vitamin C derivative asdefined in claim 1, and pharmaceutically acceptable salts thereof as anactive ingredient.
 23. A composition for skin whitening containing atleast one selected from the vitamin C derivative as defined in claim 1,and pharmaceutically acceptable salts thereof as an active ingredient.24. A method of stabilizing vitamin C, comprising the steps of:introducing glucose or a phosphoryl group or a phosphate derivativerepresented by Chemical Formula 6 to substitute for a hydroxyl group atthe 2′ carbon position of vitamin C; binding a peptide molecule having 3to 10 amino acid residues selected from the group consisting of glycine,lysine, histidine, serine, proline, hydroxyproline, threonine, glutamicacid, methionine, glutamine, and arginine, to 5′ or 6′ carbon positionof vitamin C through a dicarboxylic acid represented by Chemical Formula8:

wherein Y is chlorine, bromine, fluorine, or iodine, and m is an integerbetween 2 and
 5. 25. The method according to claim 24, wherein thepeptide molecule is selected from glycine-histidine-lysine,glycine-lysine-histidine, glycine-proline-hydroxyproline,lysine-threonine-threonine-lysine-serine, and glutamic acid-glutamicacid-methionine-glutamine-arginine-arginine.